The Himalayan mountain range has played a dual role in shaping the genetic landscape of the region by (1) delineating east-west migrations including the Silk Road and (2) restricting human dispersals, especially from the Indian subcontinent into the Tibetan plateau. In this study, 15 hypervariable autosomal STR loci were employed to evaluate the genetic relationships of three populations from Nepal (Kathmandu, Newar and Tamang) and a general collection from Tibet. These Himalayan groups were compared to geographically targeted worldwide populations as well as Tibeto-Burman (TB) speaking groups from Northeast India. Our results suggest a Northeast Asian origin for the Himalayan populations with subsequent gene flow from South Asia into the Kathmandu valley and the Newar population, corroborating a previous Y-chromosome study. In contrast, Tamang and Tibet exhibit limited genetic contributions from South Asia, possibly due to the orographic obstacle presented by the Himalayan massif. The TB groups from Northeast India are genetically distinct compared to their counterparts from the Himalayas probably resulting from prolonged isolation and/or founder effects.