Malana, a small village in Kullu District of Himachal Pradesh, India, has historically been considered a hermit village. Today it has a census size of 1,101 individuals. Geographic, linguistic, and population barriers have contributed to its seclusion. Little is known about the extent to which the population genetically differentiated during the years of isolation. To address this issue, we genotyped 48 Malani individuals at 15 highly polymorphic autosomal STR loci. We found that Malanis have lost some genetic variability compared with the present-day cosmopolitan caste populations and highly mobile pastoral cultures of India. But there is no evidence that they have gone through a severe bottleneck; the average population still shows a mean of 6.86 alleles per locus compared to a mean of 7.80–8.93 for nonisolated populations. An analysis of molecular variance (AMOVA) differentiates Malanis from the rest of the comparison populations. The population structure revealed by multidimensional scaling analysis of standard genetic distances lends support to the AMOVA. Our results are consistent with the social heterogeneity of the Malanis. We also analyzed 17 Y-chromosome STRs in 30 individuals to assess the paternal gene pool. The Malanis are characterized by a generally low Y-chromosome haplotype diversity. A network analysis indicates that two closely related haplotypes account for a large proportion of Malani Y chromosomes. We predicted Y-chromosome haplogroups and found that J2 and R1a were the most prevalent. Genetic drift and limited gene flow leading to reduced genetic diversity were important in determining the present genetic structure of the highly endogamous Malana village.