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Calotes versicolor Daudin, 1802, is geographically widespread along the Mekong River basin. The Mekong River is play important role as a significant natural barrier to several terrestrial animals living on different sides. This study aims to analyze the genetic diversity and population structure of C. versicolor populations collected from different sides of Mekong River using mitochondrial cytochrome c oxidase subunit 1 (CO1) sequences. We obtained sequences of 200 individuals from 18 sampling localities from left and right sides of the Mekong River in Lao PDR and Thailand respectively. Overall, 91 haplotypes were detected, which reflect high levels of genetic diversity in this species at the study areas. Haplotype network and phylogenetic analyses revealed that there were six major lineages (lineage C – lineage H) of C. versicolor populations within the Mekong River, whereas lineages A and B have previously been found from China and Vietnam. The genetic distance among C. versicolor was significantly related to spatial distance, however, the Mekong River had no significant effect on genetic distance. Our findings, together with previous studies, suggests that C. versicolor in Asia is a species complex with other cryptic lineages being likely but there is a need for further exploration. Thus, comprehensive genetic, biological and ecological studies of C. versicolor should be conducted throughout its entire distribution range.