The tick, Rhipicephalus microplus is considered as the most notorious ectoparasite of veterinary importance in tropical and sub-tropical regions of the world. The present study deals with the molecular characterization of R. microplus in different regions of Andaman and Nicobar Islands using sequence information of mitochondrial cytochrome C oxidase subunit I (COX1) and their phylogenetic relationship with other Indian R. microplus genotypes. DNA polymorphism study identified a total of eight haplotypes with haplotypediversity of 0.909 ± 0.065 and nucleotide diversity of 0.01911 ± 0.00493. Currently, R. microplus complex consists of five taxa; R. microplus clade A sensu Burger et al. (2014), R. microplus clade B sensu Burger et al. (2014), R. microplus clade C sensu Low et al. (2015), R. australis and R. annulatus. Phylogenetic analysis revealed the presence of two clades (clade A and clade C) of R. microplus in Andaman and Nicobar isolates; Nicobar isolates belonged to clade A whereas Andaman isolates belonged to clade C of R. microplus complex. All the other Indian sequences retrieved from GenBank belonged to clade C of R. microplus complex. Andaman isolates under clade C of R. microplus were phylogenetically distinct from Indian isolates, which indicates independent speciation under isolated island milieu. In Indian isolates, no host-specific or geographical location-specific sub-clustering was observed which indicates the species jumping potential of the R. microplus tick. Therefore, this study indicated the presence of two different genetic makeup of R. microplus complex in two areas of the Andaman and Nicobar archipelago separated by a natural geographical barrier. This indicates presence of two different founding populations of ticks, one in the south and north-middle Andaman and the other in Nicobar Island.