ABSTRACT The present study examined the genetic diversity of two populations of Dotilla myctiroides (Milne-Edwards) from Peninsular Malaysia. Based on 640-bp COX1 (cytochrome c oxidase subunit 1) gene sequences, the D. myctiroides specimens from Peninsular Malaysia, Singapore and India formed a lineage which was distinctly separated from Dotilla wichmanni and D. fenestrata. Sixteen COX1 haplotypes were found in Peninsular Malaysia samples, with 11 haplotypes in the Morib sample, eight in the Tanjung Tuan sample, and three common haplotypes in these samples. The Singapore specimen had the commonest haplotype of Peninsular Malaysia, while the Indian specimen had a distinct haplotype. The proportions of the two most common COX1 haplotypes were not significantly different between the Morib and Tanjung Tuan samples. The difference in the mean haplotype diversity between these samples was statistically significant. However, the nucleotide diversity of the two samples was not significantly different and there was no genetic subdivision/differentiation between the two populations. The high number of closely related haplotypes indicates the possibility of a recent population expansion. The data provide valuable information for future studies on the genetic diversity, population structure and phylogeography of Dotilla species.