Leucinodes orbonalis is the most detrimental South and Southeast Asian insect pest of eggplant. To help reduce the impact of this pest, population genetic diversity and structure of L. orbonalis were examined in eight populations from six countries using mitochondrial cytochrome c oxidase subunit I DNA sequences. No correlation between genetic diversity and geographic distance was detected among populations. Low levels of haplotype and nucleotide diversities were observed in the Philippines population, suggesting recent colonization. No significant gene flow was found among local populations in different countries. The Vietnam population is highly differentiated, indicated by significant pairwise FST values, and may be ascribed to a new subspecies or race. India was confirmed to be the source of genetic variation in L. orbonalis populations. Our study showed that L. orbonalis formed subpopulations for each local region, and the corresponding pest management technology should be developed at the country scale.