Simple SummaryThe geckos of the genus Uroplatus include peculiar endemic species to Madagascar. Even though they have been the subject of several morphological and molecular studies, karyological analyses have been performed only on U. phantasticus, leaving the chromosomal diversity of the genus completely unexplored. In this study, we performed a preliminary molecular analysis and a comparative cytogenetic study providing the first karyotype description of eight species of Uroplatus and an assessment of their karyological variability. We found chromosome diversity in the species studied in terms of total chromosome number (2n = 34–38), localization of loci of Nucleolar Organizer Regions (NORs) (alternatively on the 2nd, 6th, 10th or 16th pair), heterochromatin composition and occurrence of heteromorphic sex chromosome pairs. Adding our newly generated data to those available from the literature, we show that in the genus Uroplatus, as well as in a larger group of phylogenetically related gecko genera, chromosome diversification mainly occurred toward a reduction in the chromosome number by means of chromosome fusions and translocation of NOR-bearing chromosomes. We also hypothesize that the diversification of sex chromosome systems occurred independently in different genera.We provide here the first karyotype description of eight Uroplatus species and a characterization of their chromosomal diversity. We performed a molecular taxonomic assessment of several Uroplatus samples using the mitochondrial 12S marker and a comparative cytogenetic analysis with standard karyotyping, silver staining (Ag-NOR) and sequential C-banding + Giemsa, +Chromomycin A3 (CMA3), +4′,6-diamidino-2-phenylindole (DAPI). We found chromosomal variability in terms of chromosome number (2n = 34–38), heterochromatin composition and number and localization of loci or Nucleolar Organizer Regions (NORs) (alternatively on the 2nd, 6th, 10th or 16th pair). Chromosome morphology is almost constant, with karyotypes composed of acrocentric chromosomes, gradually decreasing in length. C-banding evidenced a general low content of heterochromatin, mostly localized on pericentromeric and telomeric regions. Centromeric bands varied among the species studied, resulting in CMA3 positive and DAPI negative or positive to both fluorochromes. We also provide evidence of a first putative heteromorphic sex chromosome system in the genus. In fact, in U. alluaudi the 10th pair was highly heteromorphic, with a metacentric, largely heterochromatic W chromosome, which was much bigger than the Z. We propose an evolutionary scenario of chromosome reduction from 2n = 38 to 2n = 34, by means of translocations of microchromosomes on larger chromosomes (often involving the NOR-bearing microchromosomes). Adding our data to those available from the literature, we show that similar processes characterized the evolutionary radiation of a larger gecko clade. Finally, we hypothesize that sex chromosome diversification occurred independently in different genera.