The taxonomy of the Malagasy leaf-tailed geckos Uroplatus fimbriatus and U. giganteus is in need of revision since a molecular study casted doubt on the species status of U. giganteus from northern Madagascar. In this study we separately analyse DNA sequences of a mitochondrial gene (12S rRNA) and of four nuclear genes (CMOS, KIAA1239, RAG1, SACS), to test for concordant differentiation in these independent markers. In addition to the molecular data we provide a comprehensive review of colour variation of U. fimbriatus and U. giganteus populations from the entire distribution area based on photographs. The molecular evidence clearly supports a two-species taxonomy, with U. fimbriatus corresponding to a southern clade and U. giganteus to a northern clade. This conclusion relies on the high mitochondrial divergence among these units, and especially on the full concordance of the mitochondrial signal with differentiation in two of the four nuclear genes, which show no haplotype sharing among the northern and southern clade. This suggests limited or absent gene flow among these units, even in areas where they occur in rather close proximity to one another. The only consistent difference in colour variation between the two species is the whitish iris with rather indistinct brown vertical lines in U. giganteus, versus a yellowish iris with more distinct, continuous lines of typically a more reddish brown tone in U. fimbriatus. In the U. giganteus population of Montagne d’Ambre, a particular colour pattern on the head prevails which is not present in all the specimens studied from Marojejy. The Marojejy population of U. giganteus is also genetically distinct, as is one sample from Ankavanana included in a previous study, requiring in-depth future study.