In the queenless ant genus Diacamma, one mated worker (called gamergate) maintains reproductive mono-poly in a colony by mutilating newly emerged workers. However, in several populations from south India, referred to as nilgiri, gamergates do not mutilate their nestmates but monopolize reproduction using dominance interactions . Various lines of evidence indicate that nilgiri populations are closely related to the neighboring species D. ceylonense. To determine whether this important behavioural difference between nilgiri and D. ceylonense is associated with signi-ficant genetic differentiation, we have used microsatellite and mitochondrial markers to examine genetic variation within and between nilgiri and D. ceylonense. We found a very high genetic differentiation between the two forms, which suggests a lack of gene flow. There was an unexpected pattern of mitochondrial variation, because all nilgiri populations show identical or very closely related COII sequences except one population with a very different haplotype. This divergent haplotype is genetically much more distant from the other nilgiri haplotypes than are D. ceylonense haplotypes. This pattern is not observed at the nuclear level, which suggests that introgression of mitochondrial DNA probably occurred in some nilgiri populations.