Background: Y-chromosome-specific short tandem repeat markers reside on the non-recombinant portion of the Y chromosome and are paternally inherited. These properties make Y-STRs a useful tool in investigations of sexual assault and other violent crimes, paternity cases, genealogical testing, and evolutionary studies. Native populations reside in the Peruvian coast, mountains, and jungle; however, the Mestizos are the largest population group in Peru. Results: Of the 290 samples, 283 distinct haplotypes were observed. Gene diversities for each Y-STR marker ranged from 0.4271 to 0.8597. The haplotype diversity for the total sample was 0.9964. Conclusions: Genetic affinity was identified between the Peruvian Mestizo population and other Peruvian populations (except Jivaro, Awajun, and Shipibo-Conibo populations) and Bolivian Mestizo populations using multidimensional scaling and phylogenetic tree analysis.