Black-spotted and red-spotted tokay geckos are distributed in different regions and have significant differences in morphological appearance, but have been regarded as the same species, Gekko gecko, in taxonomy. To determine whether black-spotted and red-spotted tokay geckos are genetically differentiated, we sequenced the entire mitochondrial cytochrome b gene (1147 bp) from 110 individuals of Gekko gecko collected in 11 areas including Guangxi China, Yunnan China, Vietnam, and Laos. In addition, we performed karyotypic analyses of black-spotted tokay geckos from Guangxi China and red-spotted tokay geckos from Laos. These phylogenetic analyses showed that black-spotted and red-spotted tokay geckos are divided into two branches in molecular phylogenetic trees. The average genetic distances are as follows: 0.12–0.47% among six haplotypes in the black-spotted tokay gecko group, 0.12–1.66% among five haplotypes in the red-spotted tokay gecko group, and 8.76–9.18% between the black-spotted and red-spotted tokay geckos, respectively. The karyotypic analyses showed that the karyotype formula is 2n = 38 = 8m 2sm 2st 26t in red-spotted tokay geckos from Laos compared with 2n = 38 = 8m 2sm 28t in black-spotted tokay geckos from Guangxi China. The differences in these two kinds of karyotypes were detected on the 15th chromosome. The clear differences in genetic levels between black-spotted and red-spotted tokay geckos suggest a significant level of genetic differentiation between the two.