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Background: We have examined genetic diversity at fifteen autosomal microsatellite loci in seven predominant populations of Orissa to decipher whether populations inhabiting the same geographic region can be differentiated on the basis of language or ancestry. The studied populations have diverse historical accounts of their origin, belong to two major ethnic groups and different linguistic families. Caucasoid caste populations are speakers of Indo-European language and comprise Brahmins, Khandayat, Karan and Gope, while the three Australoid tribal populations include two Austric speakers: Juang and Saora and a Dravidian speaking population, Paroja. These divergent groups provide a varied substratum for understanding variation of genetic patterns in a geographical area resulting from differential admixture between migrants groups and aboriginals, and the influence of this admixture on population stratification. Results: The allele distribution pattern showed uniformity in the studied groups with approximately 81% genetic variability within populations. The coefficient of gene differentiation was found to be significantly higher in tribes (0.014) than caste groups (0.004). Genetic variance between the groups was 0.34% in both ethnic and linguistic clusters and statistically significant only in the ethnic apportionment. Although the populations were genetically close (FST = 0.010), the contemporary caste and tribal groups formed distinct clusters in both Principal- omponent plot and Neighbor-Joining tree. In the phylogenetic tree, the Orissa Brahmins showed close affinity to populations of North India, while Khandayat and Gope clustered with the tribal groups, suggesting a possibility of their origin from indigenous people. Conclusions: The extent of genetic differentiation in the contemporary caste and tribal groups of Orissa is highly significant and constitutes two distinct genetic clusters. Based on our observations, we suggest that since genetic distances and coefficient of gene differentiation were fairly small, the studied populations are indeed genetically similar and that the genetic structure of populations in a geographical region is primarily influenced by their ancestry and not by socio-cultural hierarchy or language. The scenario of genetic structure, however, might be different for other regions of the subcontinent where populations have more similar ethnic and linguistic backgrounds and there might be variations in the patterns of genomic and socio-cultural affinities in different geographical regions. [ABSTRACT FROM AUTHOR]