Y chromosome short tandem repeat (Y-STR) loci are important genetic markers for forensic biological evidence analyses. However, paternal inheritance, reduced effective population size, and lack of independence between loci can reduce Y-STR diversity and may yield greater population substructure effects on a locus-by-locus basis compared with the autosomal STR loci. Population studies are necessary to assess the genetic variation of forensically relevant markers so that proper inferences can be made about the rarity of DNA profiles. This study examined 16 Y-STRs in three sampled populations of Native Americans from Alaska: Inupiat, Yupik, and Athabaskan. Population genetic and statistical issues addressed were: (1) the degree of diversity at locus and haplotype levels, (2) determination of the loci that contribute more so to haplotype diversity, and (3) the effects of population substructure on forensic statistical calculations of the rarity of a Y-STR profile. All three population samples were highly polymorphic at the haplotype level for the 16 Y-STR markers; however, the Native Americans demonstrated reduced genetic diversity compared with major US populations. The degree of substructure indicated that the three populations were related and admixed in terms of paternal lineage. The examination of more polymorphic loci may be needed to increase the power of discrimination of Y-STR systems in these populations.