For the development of 19-plex Y STR system and polymorphism studies in local ethnic populations sixteen markers of non-recombining regions (NRY) of Y chromosome, which show high power of discrimination among individuals, were selected in this study. Blood samples (600) were collected from the males of three most common castes of Pakistani population (Arain, Awan and Rajput) with different parent lineages. Three markers (DYS385a/b, DYS389I/II and YCAIIa/b) among 16 Y STRs are double-targeted regions of the Y chromosome and thus provide two polymorphic peaks for each respective primer set. These 16 Y-STRs were developed into Megaplex system for simultaneous amplification of all markers within the population. The overall power of discrimination observed in focused populations was 60.5%, 66.5% and 55% in Rajput, Awan and Arain casts respectively. This discrimination power will be helpful in human identification for forensic casework studies including sexual assaults and paternity testing. For the development of 19-plex Y STR system and polymorphism studies in local ethnic populations sixteen markers of non-recombining regions (NRY) of Y chromosome, which show high power of discrimination among individuals, were selected in this study. ) were collected from the males of three most common castes of Pakistani population (Arain, Awan and Rajput) with different parent lineages. Three markers (DYS385a / b, DYS389I / II and YCAIIa / b) among 16 Y STRs are double-targeted regions of the Y chromosome and thus provide two polymorphic peaks for each respective primer set. These overall power of discerbable observed in populations was 60.5%, 66.5 % and 55% in Rajput, Awan and Arain casts respectively. This discrimination power will be helpful in human identification for forensic casework studies including sexual assaults and paternity testing.