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To investigate the genetic structure of human populations in the South-west region of Iran,mitochondrial first hypervariable DNA sequences were obtained from 50 individuals representing three different ethnic groups from Khuzestan Province.Studied groups were Shushtari Persians and Chahar Lang Bakhtiyaries from Indo-Europeanspeaking populations and Bani Torof Arabs from Semitic-speaking linguistic families.Genetic analysis of mtDNA data showed high similarity of Chahar Lang Bakhtiyaries with other Iranian Indo-European-speaking populations while Shushtaries and Bani Torofs had a closer affinity with Semitic-speaking groups rather than to other Iranian populations.The relationship of Chahar Lang Bakhtiyaries and Bani Torof Arabs with their neighbor populations can be explained by linguistic and geographic proximity.Whereas,the greater similarity of Shushtari Persians with West Asian Arabs is probably according to high gene flow between them.This article represents a preliminary study of three major ethnic groups of South-west Iran which investigates the potential genetic substructure of the region.