Andaman Negrito mtDNA lineages survived in complete isolation from South and Southeast Asians since out-of-Africa. Autosomal STR markers show high polymorphism and 77% mean gene diversity in Great Andamanese, Onge, and Nicobarese. Andaman Negrito populations lack affinities with Africans or Indians confirming unique origin. Nicobarese show close genetic affinities with Southeast Asians indicating recent entry. Great Andamanese and Onge represent isolated Negrito lineages. High within-population heterozygosity from 0.392 to 0.857. Allele range 7-31.2 across markers. No shared ancestry with mainland Indian populations. Negritos diverged early post-out-of-Africa settlement. Nicobarese distinct from Negritos genetically.