Chameleons are well-known, highly distinctive lizards characterized by unique morphological and physiological traits, but their karyotypes and sex determination system have remained poorly studied. We studied karyotypes in six species of Madagascan chameleons of the genus by classical (conventional stain, C-banding) and molecular (comparative genomic hybridization, in situ hybridization with rDNA, microsatellite, and telomeric sequences) cytogenetic approaches. In contrast to most sauropsid lineages, the chameleons of the genus show chromosomal variability even among closely related species, with diploid chromosome numbers varying from 2 = 22 to 2 = 28. We identified female heterogamety with cytogenetically distinct Z and W sex chromosomes in all studied species. Notably, multiple neo-sex chromosomes in the form ZZZZ/ZZW were uncovered in four species of the genus (, , and previously studied ). Phylogenetic distribution and morphology of sex chromosomes suggest that multiple sex chromosomes, which are generally very rare among vertebrates with female heterogamety, possibly evolved several times within the genus . Although acrodontan lizards (chameleons and dragon lizards) demonstrate otherwise notable variability in sex determination, it seems that female heterogamety with differentiated sex chromosomes remained stable in the chameleons of the genus for about 30 million years.
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