Sexual assault is a violent crime and a significant problem faced by any developing society. Identification of spermatozoa is the biological evidence most often sought in specimens from sexual assault victims. Absence of spermatozoa usually terminates biological investigations, and the victim’s testimony is contested. DNA with high discrimination potential and sensitivity obtained in an unbiased manner would be highly advantageous in sexual assault cases with negative results. Cervico-vaginal, oral and anal swabs were collected from 930 sexual assault cases, within a period from July 2006 to December 2007. In a total of 930 cases, 300 cases (32.25%) were found cytologically negative. Y-Chromosomes were detected in 108/300 (36%) cases, a fairly high percentage indeed for victims whose testimony would have been contested otherwise. Y-chromosome was also evidenced in the population of victims examined after 72 hours of sexual assault. Spermatozoa are rarely detected at such intervals, so medical men are reluctant to take samples, but our studies show that swabs ought to be taken from victims for Y-Chromosome DNA typing even after long lapses of time between sexual assaults and medical examination. Y- STRs are also very useful in detecting the male DNA fraction in male/ female DNA mixture where amount of female DNA is present in a far excess amount. It can also provide evidence in old and wasted samples. Eleven Y-STR(short tandem repeats), namely DYS 19, DYS385 a,b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS438, DYS439, residing on the Y- chromosome and amelogenin recognized by an international body “The Scientific Working Group on DNA Analysis Methods” (SWGDAM) were chosen and amplified simultaneously using polymerase chain reaction (PCR) by the Y-PLEX T M 12 system, asensitive, valid, reliable, and robust multiplex system. Amelogenin provided results for gender identification and served as internal control for PCR. It did not adversely affect the amplification of Y-STRs in mixture samples containing male and female DNA. After assurance of quality standards in accordance with the instructions laid down by DNA advisory Board (DAB), population data from Punjab was collected and comparison made with studies from other global population groups in addition to 12 ethnic groups from Pakistan. It showed greater genetic similarity with Africa, West Asia, and Europe but not with China & Japan. Arlequin version 3.0, a standard software for haplotyping analysis was used, the value of haplotype diversity is 0.9766 +/- 0.0074, gene diversity 0.67619 with standard deviation 0.10293. The value of average diversity over loci is 0.672759 +/- .354019. The mean number of pair wise differences are 6.727587 +/- 3.196717. Therefore, after adopting a proper strategy of designing and optimising multiplex system for Y-STRs, we can successfully resolve the matters of individual identification, determination of perpetrators of violent sexual crime, of unestablished paternity and paternal lineage.