Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the most pathogenic and economically significant pathogens of poultry. The studies were designed to compare and evaluate conventional as well as molecular diagnostic capabilities for the identification of pathogenic avian mycoplasma species; and to delineate the epidemiological factors involved in dissemination of mycoplasma infections within and across the farms environment. In this study, serology, culture isolation and polymerase chain reaction (PCR) methods were applied and compared to document the involvement of M. gallisepticum and M. synoviae infection in respiratory distress cases of chickens. Rapid serum agglutination test (RSA) was applied to determine sero-prevalence of MG and MS. Culture isolation was made on modified Frey’s medium. PCR assays based on detection of 16SrRNA gene of M. gallisepticum and M. synoviae were applied to confirm identification of species. Conventional duplex PCR was optimized to amplify the non- conserved genes like the cytadhesin encoding surface protein (mgc2 gene) of M. gallisepticum and a variable heamagglutinin surface protein (vlhA gene) of M. synoviae. DNA sequence analysis was made for indigenous isolates of MG and MS. Real-time PCR was developed using SYBR green as well as TaqMan procedure. Duplex TaqMan minor groove binder (MGB) real-time PCR was developed for simultaneous detection of MG (mgc2) and MS (vlhA) genes with internal positive control (IPC). Present study documented the overall sero-prevalence of MS (79.55%), MG (72.89%) and MG/MS mix infections (19.20%) as determined through RSA test. The sero-positive cases further resolved the culture recovery of MG (71.67%), MS (49.69%) and MG/MS mix infections (32.35%). Amplicon size polymorphism was observed for indigenous isolates of MG as 288bp amplicon size, which was found different as compared to known strains reported so far. DNA sequence was submitted to GenBank as M. gallisepticum (mgc2 gene) strain Egpk1UAF08 partial sequence, (GenBank accession No. FJ395202). For M. synoviae with amplicon size of 373bp, the amplicon size polymorphism was not observed and the DNA sequence was submitted as M. synoviae (vlhA gene) strain Espk1UAF08, (GenBank accession No. FJ409871). xiiDuplex conventional PCR was efficiently optimized for the simultaneous detection of MG/MS at 288bp/373bp. Specimens collected from sero-positive cases successfully accomplished the prevalence (determined through PCR) for MS (98.14%), followed by MG (93.34%) and MG/MS combined infection (82.35%). The sensitivity of PCR method was calculated as 100% for MG and MS. The overall results indicated reciprocal increase in PCR detection frequency of MG and MS from flocks with the increase in sero-positivity while, the increase in the sero-prevalence has resulted into a significant decrease in culture isolation of MS but for MG. Prevalence of MG based on (RSA+Culture) results was ranked highest in broiler (76.66%), followed by layer (75.07%) and breeder (62.88%) flocks. Prevalence of MS (RSA+Culture) was found maximum in layer (80.86%), followed by breeder (80%) and broiler (76.57%) birds. Moreover, the true prevalence calculation was based on PCR results obtained for sero- positive samples. The true prevalence of MG (RSA+PCR) was found highest in broiler (96.82%), followed by layer (91.90%) and breeder (88.19%) flocks. The true prevalence of MS (RSA+PCR) was found highest in broiler (98.82%), followed by breeder (98.11%) and layer (97.84%). On the basis of successful PCR results obtained from the specimens, the priority of selection for MG detection include tracheal swab followed by nasal, lung, oral, air sac, cloacal and synovial fluid. Similarly, the preferred specimens for MS detection were included as tracheal swab followed by synovial fluid, nasal, oral, air sac, lung and cloacal swab. The most frequent precipitating factors found to contaminate the farm environment with MG belonged to egg shell (50%) followed by farm dust (35%) and least from feathers and incubator (15%). Whereas, the most frequent contaminating environmental specimens for MS were recorded as egg shells (35%), followed by drinkers (30%) and feathers (5%). The SYBR green real-time PCR was optimized for the detection of 16SrRNA gene with average melting temperature (Tm) of 80.5°C at mean cycle threshold (Ct) value of 14.7 cycles for M. gallisepticum; and average Tm of 80.7°C at mean Ct value of 14.7 cycles for M. synoviae. The mgc2 gene based SYBR green real-time PCR for MG was optimized with an average Tm of 81.5 o C at mean Ct values of 14.5 cycles. New SYBR green real-time PCR assays were developed and optimized based on the primers designed xiiifrom indigenous DNA sequence of (mgc2 gene) at average Tm of 73.5 o C at mean Ct value of 15.5 cycle for M. gallisepticum; and indigenous DNA sequence of (vlhA gene) with average Tm of 76.0°C at mean Ct value of 16.2 cycles for M. synoviae. Moreover, the SYBR green real-time PCR assays were upgraded to TaqMan MGB probe based assays. In TaqMan 6FAM labeled MGB probe based Plus-Minus assay procedure, the Ct values were observed between (14-30.2) cycles for the positive samples tested for MG. Plus-Minus assay for MS, showed a minimum Ct value of 19 cycles for the tested samples. Real-time PCR products were successfully cloned into pCR2.1-TOPO cloning vector and the successful clones were selected. TaqMan real-time PCR based absolute quantification has indicated an inverse linear relationship between Ct values and the target DNA concentration. For Duplex TaqMan MGB real-time PCR reaction the maximum sensitivity of MG probe (6FAM labeled) was found at mean Ct value of 15.5 cycles. Whereas, for MS probe (NED labeled), the mean Ct value of 20.5 cycle was observed. In conclusion, the PCR based detection of M. gallisepticum and M. synoviae infection was useful because of the time and difficulties associated in obtaining pure cultures. Also, it is suggested that, the true prevalence of M. gallisepticum and M. synoviae may best be reflected by combining PCR results with RSA test findings. The present study has documented the involvement of indigenous strains of MG and MS in the respiratory distress cases of chickens and farm environment using duplex PCR assays. The DNA sequence analysis of indigenous mycoplasma strains deposited to GenBank may help to further elucidate the molecular epidemiology of MG and MS infection for an outbreak investigation in and across the country. Also, the TaqMan ® MGB probe based duplex real-time PCR developed for simultaneous detection of MG (mgc2 gene) and MS (vlhA gene), along with TaqMan Exo (Exogenous internal positive control) IPC, was proved simple, robust, more specific and cost effective alternative to previously described methods, and will particularly be beneficial for high-throughput diagnostic laboratories.