The aboriginal tribal groups living in the Andaman and Nicobar Islands are thought to be the descendants of people who were part of the early human dispersal into the Southeast Asia. However, the origin of the tribes of Andaman, the region from where the aboriginals arrived and the route of their migration are still a matter of great speculation. To explore the origin and affinities of the Andaman Islanders, we studied the polymorphism at fifteen autosomal short tandem repeat (STR) loci, mitochondrial control region sequences and eight Y chromosomal STR loci in 194 blood samples, of which 94 samples were from the Andaman Negritos (24 Great Andamanese and 70 Jarawas) and 100 Nicobarese, a Mongoloid group of Nicobar Island and evaluated their relatedness with similar ethnic groups of India, Southeast Asia and Africa. Our results clearly demonstrate that the aboriginal populations of the Andaman Islands – the Great Andamanese and the Jarawas constitute an independent cluster, separating out from all other populations selected in the study. The Nicobarese show a close affinity with the Mongoloid population of Southeast Asia. The distinct genetic identity of the aboriginal populations of the Andaman Islands and other Asian and African populations deciphered by nuclear and mitochondrial DNA diversity suggest that (i) either the aboriginals of Andaman are one of the surviving descendents of settlers from an early migration out of Africa who remained in isolation in their habitat in Andaman Islands, or (ii) they are the descendents of one of the founder populations of modern humans.