摘要This study was performed to explore the genetic diversity and genetic structure of red-spotted tokay geckos (Gekko gecko) from 23 different geographical areas in Thailand, Lao PDR and Cambodia. The mitochondrial tRNA-Gln/tRNA-Met/partial NADH dehydrogenase subunit 2 from 166 specimens was amplified and sequenced. A total of 54 different haplotypes were found. Highly significant genetic differences occurred between populations from different localities. The haplotype network revealed six major haplogroups (G1 to G6) belonging to different clades (clade A–E). Clade D and clade E were newly observed in this study. Haplogroup G4 (clade D) was a sympatric population with haplogroup G1 (clade B). The populations from northern Thailand were divided into two distinct haplogroups separated by mountain range. Genetic structure and genetic differentiation of the tokay in Southeast Asia was related to the geographical region sampled, spatial distance and natural barriers. Our results indicate that red-spotted tokay geckos from mainland Southeast Asia are cryptically diverse. Morphological comparisons, in addition to an intensive genetic investigation covering the whole species range, are needed to clarify the systematic and population structure of this species group.