The genus Gekko comprises a diverse group of nocturnal lizards that are widely distributed across the plains and plateaus of temperate and tropical Asia, as well as the western islands of the Pacific Ocean. In this study, mitochondrial DNA and morphological data were integrated to diagnose and describe a novel species, Gekko wuzhengjuni sp. nov., from Guilin, Guangxi Zhuang Autonomous Region, China. Phylogenetic analyses indicate that this species forms a monophyletic lineage that is sister to G. fengshanensis within the Japonigekko subgenus, as determined by mitochondrial 16S rRNA (16S) and NADH dehydrogenase subunit 2 gene (ND2) sequences. The uncorrected genetic distances for mitochondrial 16S rRNA sequence divergence range from 9.85% (G. liboensis) to 19.11% (G. subpalmatus), whereas ND2 divergence ranges from 14.62% (G. fengshanensis) to 32.84% (G. liui) relative to other congeners. Morphologically, Gekko wuzhengjuni sp. nov. can be distinguished from its congeners in the region by a specific combination of characteristics: (1) nares that contact the rostral scale, with the absence of an internasal scale; (2) the presence of two enlarged postmental scales; (3) enlarged tubercles extending from the area posterior to the eyes along the neck to the base of the tail, arranged in eight or nine rows at midbody; (4) a total of 140–143 midbody scale rows; (5) precloacal pores continuous, numbering 10 in males and absent in females; and (6) a single postcloacal tubercle present on each side.