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Regional variation in the genetic constitution and genetic structure of the populations of the Kinnaur district, Himachal Pradesh, India, was examined from the data of 9 blood group, 11 red cell enzyme and 4 serum protein loci. Not only the two main ethnic groups—Kanet and Koli—showed genetic differences, but also the Kanet population itself was found to be genetically heterogeneous. The Kanet from the Puh region appears to be distinct. There was sufficient gene differentiation among the regional subpopulations to allow an overall excess of homozygosity. The mean FIS value for each locus was found consistently higher than FST, suggesting that the infrastructure of the populations of Kinnaur is highly influenced by inbreeding. Various measures of genetic distance show consistent results with each other. There is no evidence that selection has any appreciable effect on the local differentiation, but the analysis suggests that the differences are more likely to be due to inbreeding, admixture and ethnic affiliation.