Linguistic and ethnic diversity throughout the Himalayas suggests that this mountain range played an important role in shaping the genetic landscapes of the region. Previous Y-chromosome work revealed that the Himalayas acted as a biased bidirectional barrier to gene flow across the cordillera. In the present study, 17 Y-chromosomal short tandem repeat (Y-STR) loci included in the AmpFlSTR® Yfiler kit were analyzed in 344 unrelated males from three Nepalese populations (Tamang, Newar, and Kathmandu) and a general collection from Tibet. The latter displays the highest haplotype diversity (0.9990) followed by Kathmandu (0.9977), Newar (0.9570), and Tamang (0.9545). The overall haplotype diversity for the Himalayan populations at 17 Y-STR loci was 0.9973, and the corresponding values for the extended (11 loci) and minimal (nine loci) haplotypes were 0.9955 and 0.9942, respectively. No Y-STR profiles are shared across the four Himalayan collections at the 17-, 11-, and nine-locus resolutions considered, indicating a lack of recent gene flow among them. Phylogenetic analyses support our previous findings that Kathmandu, and to some extent Newar, received significant genetic influence from India while Tamang and Tibet exhibit limited or no gene flow from the subcontinent. A median-joining network of haplogroup O3a3c-M134 based on 15 Y-STR loci from our four Himalayan populations suggests either a male founder effect in Tamang, possibly from Tibet, or a recent bottleneck following their arrival south of the Himalayas from Tibet leading to their highly reduced Y single-nucleotide polymorphism and Y-STR diversity. The genetic uniqueness of the four Himalayan populations examined in this study merits the creation of separate databases for individual identification, parentage analysis, and population genetic studies.