Available mitochondrial DNA sequences of Cytochrome oxidase subunit – I gene of assassin bugs were subjected to phylogenetic analysis to understand the intergeneric and interspecific variations and the role of geographical isolation on speciation; using CLUSTAL W in MEGA version 5.1. This analysis includes twenty five species belonging to fourteen genera from five countries viz., India, USA, China, Brunei and South Korea. The pairwise genetic distances were calculated and phylograms were constructed using Maximum Likelihood, Maximum Parsimony, and Neighbor-Joining methods. This analysis revealed phylogenetic relationships and the role of geographical isolation on speciation.