Simple SummaryWe implemented a molecular and phylogenetic analysis and a comparative karyological investigation with standard and chromosome banding methods on different taxa of the endemic Malagasy velvet geckos of the genusBlaesodactylus. We present the description of the karyotype of three different taxa and a characterization of the chromosomal diversity in the genus. We show the occurrence of karyological variability in the genusBlaesodactylusin terms of chromosome number (2n = 40–42), morphology, chromosome position of loci of NORs, and distribution pattern of heterochromatin. Considering our results together with the available information on evolutionary related gecko species, we hypothesize that the karyotype evolution inBlaesodactylusmostly involved a decrease in the total number of chromosomes and the formation of biarmed elements. We also highlight that similar pathways of chromosomal rearrangements have been previously observed in other geckos, possibly representing a convergent karyotype evolution.AbstractWe performed a molecular and phylogenetic analysis and a comparative cytogenetic study with standard karyotyping, silver staining (Ag-NOR) and sequential C-banding + Giemsa, + fluorochromes on severalBlaesodactylussamples. The phylogenetic inference retrieved two main clades, the first comprisesB. victori,B. microtuberculatusandB. boivini, while the second includesB. sakalava,B. antongilensisandB. ambonihazo. The available samples ofB. sakalavaform two different clades (here namedB. sakalavaclade A and clade B), which probably deserve a taxonomic re-evaluation. We found a karyological variability inBlaesodactylusin terms of chromosome number (2n = 40–42), morphology, location of NORs, and heterochromatin distribution pattern.Blaesodactylus antongilensisandB. sakalavaclade A and B showed a karyotype of 2n = 40 mostly telocentric chromosomes. Pairs 1 and 6 were metacentric inB. sakalavaclade A and B, while pair 1 was composed of subtelocentric/submetacentric elements inB. antongilensis.In contrast,B. boivinidisplayed a karyotype with 2n = 42 only telocentric chromosomes. NORs were on the first chromosome pair inB. boivini, and on the second pair inB. antongilensis. Adding our data to those available from the literature on evolutionarily related species, we highlight that the chromosome diversification in the genus probably proceeded towards a progressive reduction in the chromosome number and the formation of metacentric elements.