The aim of present study was to characterize the mitochondrial cytochrome oxidase sub unit 1 gene (COI) from Apis dorsata population inhabited at different geo-locations of southern India. A. dorsata worker bees were obtained randomly from the natural colonies in urban location, malnad, semi-arid and arid locations. Thorax was used for subsequent DNA extraction and was amplified by PCR, followed by Sanger sequencing. The mt-DNA COI nucleotides were matched with BLAST to obtain sequences from the NCBI that have high levels of homology (>90%). The gene was amplified using universal primers. Surprisingly, phylogenetic tree of A. dorsata derived from different geo-locations indicated interesting outcome. Nevertheless, the mt- COI sequences of A. dorsata derived from arid and semi-arid locations were lying on the same clade and showing 90% similarity however, mt-DNA COI sequences of A. dorsata from malnad location lies in another clade, but mt-DNA COI sequences of A. dorsata from urban location showed close relation to AY58841511-321. Moreover, the evolutionary relationship indicated 58% similarity between A. dorsata populations live in malnad and urban locations. Interestingly, the urban location has shown significant variation in nucleotide sequence compared to other geo locations based on neighbor joining and maximum likelihood methods and thus, sequence divergence existed among the A. dorsata population.