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Regional variation in the genetic constitution and genetic differentiation of three tribal populations (Koya, Lambadi, and Chenchu) of Andhra Pradesh, South India, was examined from the data of 27 polymorphic loci (9 blood groups, 13 red cell enzymes, and 5 serum proteins). Significant heterogeneity was observed among the three tribal groups at several loci (ABO, RH, P, ADA, PGM, ACP, ESD, PGD, GPT, HP, C3, and BF). Pairwise comparisons also showed significant genetic differences between the Koya and the Chenchu at seven loci, between the Koya and the Lambadi at nine loci, and between the Chenchu and the Lambadi at seven loci. Gene differentiation among the three tribes was sufficient to allow an overall excess of heterozygosity. The Fis estimates of each tribe showed positive values, but a great number of alleles showed negative FIS values, supporting varying degrees of gene flow and admixture with neighboring populations. The genetic differentiation and affinity of 14 tribal populations of Andhra Pradesh were further examined using published and unpublished data on 11 polymorphic genetic systems. Despite the genetic distinctions between two Chenchu samples and Koya and Koya-related tribes (Koya Dora and Konda Dora), geographic proximity seems to be an important determinant of affinity of the tribal populations of Andhra Pradesh. The extent of genetic diversity is high compared with previous reports from this state. No evidence from the present data indicates that selection had any appreciable effect on local differentiation, but the present analysis suggests that differences are more likely to be maintained by genetic drift, admixture, and inbreeding.