The blue-crested lizard, Calotes mystaceus Duméril & Bibron, 1837, is listed as a protected wild animal in Thailand. Its population is likely to be dramatically reduced due to massive hunting in several areas in this country. Basic information on its population genetics is therefore needed to facilitate its conservation. Thus, in this study we investigated the mitochondrial cytochrome c oxidase subunit 1 (CO1) sequence variation of 238 individualC.mystaceus from 42 different geographical localities of the north, west, central, east and northeast regions of Thailand. High genetic diversity and genetic differentiation at the intrapopulation and interpopulation levels was observed.We detected 63 unique haplotypes and 12 common/shared haplotypes. The phylogenetic analysis reveals two major lineages, I and II. These two lineages are separated by mountain ranges, which play an important role as natural barriers blocking gene flow. Our finding reveal at least two cryptic lineages represented in C. mystaceus populations in Thailand. However, a comprehensive investigation of the morphology, biology, ecology and genetic diversity of C. mystaceus in other regions within its area of distribution is needed.