Application of DNA testing has been developed into a requisite and regular component of recent forensic casework, based on highly sensitive and stable PCR techniques for the analysis of different types of biological specimens. On account of achievements accomplished in past three decades, forensic DNA analysis has become a key to convict and exonerate the suspects and to identify the victims of criminal cases, accidents and mass disasters. However there has been observed some existing imperfections and limitations in the use of conventional STR system as in conditions of highly degraded DNA, Y-amelogenin mutation, allele sharing among unrelated male individuals and complex mixtures. In the current study, development, optimization and validation of 18 loci multiplex system was accomplished comprising on 10 autosomal miniSTRs, SE33, Penta E, Penta D and four Y chromosomal STRs (DYS385a/b, DYS438 and DYS392) along with gender detection marker amelogenin into a single PCR reaction simultaneously. Amongst miniSTRs CSF1PO, D7S820, TPOX, D18S51, D13S317, FGA, D5S818, D21S11 and D16S539 have been preferred from US core loci of Combined DNA Index System (CODIS), D2S1338 selected from UK core loci (UCL) while SE33 is the part of German Core Loci database with additional two penta-nucleotide repeat markers (Penta-E and Penta-D) and Y-STRs as typically used in commercially available kits. The primer sequences were designed and synthesized in order that the final length of fragment obtained in the range of 61 bp (base pairs) to 469 bp by using 5-flourscent dyes detection system. The optimal concentration of primers was obtained in the range of 0.09-2.25 μM to get the successful amplification at each locus in a multiplex reaction. Moreover the multiplex system was validated by following SWGDAM (Scientific Working Group on DNA Analysis Methods) guidelines concerning stability, sensitivity, species specificity, accuracy and precision, DNA mixtures, population distribution genetics, forensic and paternity statistical parameters, low copy number of DNA and studies based on PCR amplification (annealing temperature, PCR conditions and PCR components). The complete DNA profile was obtained at the concentration of DNA as low as 0.125ng with 30 PCR cycles in a reduced cycling time of about one hour followed by 74% and 41% profiling results at 0.062ng and 0.031ng respectively which demonstrates the fastest detection of the system. All loci were amplified successfully except one locus using 1ng degraded DNA of up to 2 minutes treatment with Dnase-1 enzyme and 01 ng nondegraded DNA when mixed with 100ng of humic acid while amplification of 10 loci was obtained even at duration of 80 minutes enzymatic treatment. In the DNA mixtures of two and more than two contributors, the minor component obtained in the ratios of 10:1, 1:10, 10:1:1, 1:10:10, 1:10:1 and 1:1:10 was productively interpretable at threshold level of 50 RFU (relative fluorescence unit) which shows that this system is helpful in complex mixtures analysis. Population distribution studies were conducted in random individuals of major groups from two provinces of Pakistan with 337 blood samples of Punjabis and 200 blood samples of Pashtuns. A sum of 216 and 202 polymorphic alleles was observed for 17 loci among Punjabi and Pashtuns samples respectively. The SE33 was found to be the highest polymorphic and discriminatory locus among all the loci which studied in Pakistani populations. At significant probability level of p<0.003 after Bonferroni correction, no significant deviations from Hardy-Weinberg equilibrium were observed in Punjabi population while two loci (CSF1PO and TPOX) showed significant deviations in Pashtuns. The combined match probability using all 17 loci achieved up to 9.2 x 10-20 and 1.09 x 10-18 in Punjabi and Pashtun samples respectively. The probability of identity by using the product rule having the same DNA profile is 1 in quadrillions as selected randomly from the Punjabi population and 1 in quintillions for the Pashtun Population. On the basis of results obtained from validation studies along with forensic competency and parentage statistical parameters, it is demonstrated that this system will be robust and reliable to meet the existing challenges of forensic and parentage DNA analysis.