The Y chromosome microsatellite markers have been extensively used for population genetic studies and in individual identification and paternity testing in forensic medicine. In the present study, we report the data of five male-specific, polymorphic microsatellites in 740 unrelated male individuals from 12 different ethnic groups of Pakistan. The overall diversities of these individual loci in Pakistan ranged from 0.236 to 0.799. The total haplotypes identified were 152, and of these, 70 different haplotypes were present in only single individuals. Two haplotypes were found more frequently, 9_8_17_11_24 (13.5%) and 9_8_17_11_25 (8.6%), showing population-specific clustering in the Mohanna and the Brahui, respectively. An overall haplotype diversity of 0.965 in Pakistan suggested a high power of discrimination for these loci. Few populations, particularly the Mohanna and the Balti, showed lower haplotype diversity values for these loci (0.662 and 0.758, respectively). This set of microsatellite loci reported in the study can be used for population genetics and forensic medicine analysis. This study also demonstrates the importance of studying haplotype distribution pattern in population genetics.