The diverse and complex/heterogeneous Pakistani population is categorized into more than 18 ethnic groups. A properly reported forensic DNA database for this seventh largest population of the world is still not available. This study contributes towards the development of a forensic DNA database of the Pakistani population comprising both autosomal short tandem repeat (STR) markers profiles and mitochondrial DNA (mtDNA) hyper-variable regions (HVRs) haplotypes. The obtained genetic data was used for phylogenetic and demographic analyses to study the structure of the Pakistani population. Additionally, the molecular diagnostic potential of the autosomal STRs was also evaluated for the detection of chromosomal aneuploidic conditions. DNA samples from 701 individuals belonging to the Punjabi, Pathan, Sindhi, Balochi and Hazara ethnic groups of Pakistan, were analyzed for fifteen short tandem repeat (STR) markers (TPOX, D2S1338, D3S1358, FGA, CSF1PO, D5S818, D7S820, D8S1179, THO1, VWA, D13S317, D16S539, D18S51, D19S433 and D21S11) included in the AmpFlSTR® Identifiler™ PCR amplification kit. Our data showed that four markers, D2S1338, D18S51, D19S433 and FGA exhibit high power of discrimination, while TPOX was the least discriminative among all studied loci. Subsequent analyses also revealed highly significant deviations from Hardy–Weinberg equilibrium at several loci in all the studied ethnic groups, which probably occurs due to frequently practiced inbreeding (consanguineous marriages) within each group. Further analyses with the clustering algorithm STRUCTURE, principal component analysis (PCA) and neighbour joining (NJ) tree did not show clear genetic differences among the five ethnic groups. However, differences were evident with Hazara ethnic group (emerged as a genetic out-group) when the analyses were performed by using the data of 783 microsatellite markers from the HGDP-CEPH panel. Most of the STR markers in the Identifiler kit are valuable forensic tools but they are insufficient for elucidating the population structure or capturing the demarcation and variation among the studied ethnic groups of Pakistan. As the STR genotype frequency data from these five studied ethnic groups did not show any remarkable differences, it is not possible to assign ethnicity to an unknown DNA sample belonging to any of these ethnic groups on the basis of the data derived from 15 STRs. This study also attempts to investigate the applicability of AmpFlSTR® Identifiler™ PCR amplification kit for quick and simultaneous diagnosis and tracing of parental source of common chromosomal aneuploidies. Samples from 74 patients with different aneuploidic conditions were evaluated for diagnostic strengths of these STR markers. Among these aneuploidic samples, 100% of the samples with autosomal trisomies were precisely detectable using Identifiler STRs, although aneuploidies involving sex chromosomes were not detectable. Parental origin of aneuploidy was traceable in 92.54% patients with autosomal trisomies, a finding that validated the diagnostic potential of 15 STR markers for the common trisomic conditions. In order to investigate mtDNA HVRs sequence variations, we evaluated the forensic potential of the three HVRs for applicability in the Pakistani population, especially in situations where nuclear DNA is degraded. For this purpose, sequence data were generated for 104 individuals belonging to the Punjabi, Pathan, Sindhi, Balochi and Hazara ethnic groups of Pakistan. The phylogenetic analysis and comparison of the sequence data indicated that the genetic diversity is 0.9901. A total of 184 polymorphic sites were observed among all samples in the HVR-I, HVR-II, HVR-III and some other part of the mtDNA. Later haplotype analysis showed the presence of 102 haplotypes. Interestingly, 100 haplotypoes were unique to a sample and thus present a high power of discrimination (99.76%) and can be promising for forensic applications in Pakistan. However the phylogenetic analyses of the mtDNA data could not yield the genetic structure of the Pakistani population. However, the screening of intergenic COII / tRNALys 9-bp deletion/insertion polymorphism in 1233 individuals from the above mentioned five ethnic groups as well as six additional ethnic groups of Pakistan (including Brahui, Burusho, Kalash, Balti, Makrani and Parsi) demonstrated Pathans as a highly heterogeneous bearing high percentages of previously called “Asia specific” 9-bp deletion (19%) and the so called European 9-bp insertion (3.8%). Overall, the 9-bp deletion was observed in 94.16% and 9-bp insertion in 0.9% samples in all of the 1233 studied samples. These data can establish more conclusive results in conjugation with the HVRs sequence data along with their global haplotype information to provide insights into phylogenetic history and genetic demographic structure of the Pakistani population. Overall this study has contributed towards the development of an ethnically categorized allele frequency database for the Pakistani population covering both the autosomal and mitochondrial DNA. In addition, Identifiler multiplex system is presented as a valuable approach for detection of many autosomal trisomic conditions.