Toxoplasma gondii is an intracellular obligate protozoan parasite which is responsible for causing toxoplasmosis. This study was conducted in district Mardan in human population for the detection and molecular characterization of T. gondii. A total of 3132 samples were collected randomly from three tehsils, and were tested through Immuno Chromatographic Test and Enzyme Linked Immunosorbent Assay. The overall epidemiology of toxoplasmosis was 34.96%. A significant difference was found in gender in which the rate of infection in females was (37.98%) as compared to males (18.91%). The highest prevalence of toxoplasmosis was observed in age group 36-45 years (48.01%), while age group of 56-65 years were having lowest prevalence of 16.96%. The overall rate of latent and fresh infection in age group 36-45 years was highest (17.24%) and (27.97%) respectively and was lowest in age group 56-65 years (6.25%) and 10.71% respectively. The prevalence was statistically significant in the literacy groups in which high infection was observed in illiterate individuals (39.40%) as compared to literate individuals (30.67%). In overall, the illiterate group was more exposed to the fresh infection and was significant because of the awareness about the disease in the educated persons. Persons who were living in the muddy homes were more exposed to the infection as compared to the people living in concrete homes. Similarly, there was no statistical significant difference in case of rural and urban areas (P –value=0.208). The surgical patients were showing high results of 37.97% as compared to the reports of non-surgical individuals (29.22%). Patients with complications were tested for the infection and was observed that there was a very high prevalence in case of heart problem patients and kidney patients. Different tehsils showed different seropositivity rates with a significant difference (Pvalue=0.00). The disease was more significant in tehsil Mardan as compared to other tehsils. Tehsil Mardan was carrying the high infection rate of 38.68% followed by 36.22% in Katlang and Takht Bhai tehsil was having 29.83% prevalence. The epidemiology of infection in middle class people was (28.24%), whereas 48.16% was detected in the lower-class individuals. The disease was more significant in low economic status population as compared to high and middle economic status population (P-value=0.00). Moreover, a total of 160 samples cat fecal samples were collected and were observed for the detection of oocysts through microscopy. 9.3% were detected positive for T. gondii oocysts whereas the prevalence rate in male was 5.76% as compared to female stray cats in which the prevalence rate was 11.11%. The positive samples were then analyzed for further confirmation through PCR and bands were visualized on 2% gel. The DNA of positive samples was further processed for sequencing. The sequence was further BLAST for finding any variation.