Amrasca biguttula biguttula (Ishida), the cotton leafhopper, is a polyphagous insect pest of Asia and Southeast Asian countries. We sequenced a mitochondrial COI gene fragment from 67 individuals of cotton leafhopper collected from 7 major cotton growing states of North, Central, and South India. Genetic divergence analysis of leaf hopper population across India confirmed the presence of single species. Thirty haplotypes, in total, were determined across different regions of India. While population from North India was dominated by single haplotype, the south and central Indian populations show dispersion of different haplotypes across the region. The neutrality test rejection for the north Indian population also suggests population expansion. The genetic differentiation and gene flow analysis together confirmed the phylogeographic structure of the A. biguttula biguttula Ishida as isolated by distance.