A new species of the Gekko (Japonigekko) japonicus group from Khammouane Province, central Laos is described based on morphological characters and molecular evidence. Morphologically, Gekko khunkhamensis sp. nov. can be distinguished from the remaining congeners by a combination of the following characters: size moderate (SVL 69.775.2 mm); nares in contact with rostral; internasals absent; postmentals enlarged; interorbital scales between anterior corners of the eyes 31 or 32; dorsal tubercles absent; ventrals between mental and cloacal slit 181185; midbody scales 127138; ventral scales 4245; subdigital lamellae on first toe 13 or 14, on fourth toe 14 or 15; tubercles on upper surface of fore and hind limbs absent; precloacal pores absent in the male and females; postcloacal tubercles 2; tubercles absent on dorsal surface of tail base; subcaudals distinctly enlarged; dorsal surface of body with five dark grey bands, which become irregular posteriorly. Genetically, the new species is placed in a clade, consisting of G. bonkowskii, G. nadenensis, G. scientiadventura, G. sengchanthavongi, and G. thakhekensis and differs from other congeners by at least 13% in terms of pairwise distance based on a fragment of the mitochondrial ND2 gene.