The genus Enyalius is composed of 11 described species inhabiting forest areas in Amazônia, Cerrado and Atlantic forest biomes. Currently, eight species with high levels of chromosome variation have been karyotyped. The study aims to characterize the karyotype of Enyalius boulengeri, with classical and molecular techniques, and improve knowledge about the karyotype evolution of the lizard genus Enyalius. The species has 2n = 36 chromosomes (8m + 4sm + 24mc), FN = 24; NORs and 18S rDNA were subtelomeric and located on chromosome pair 2. Repetitive DNA probes (CAT)10 accumulated on centromeric and terminal regions of some macrochromosomes. (GA)15 probe showed conspicuous accumulation on the pericentromeric region of chromosome pairs 1 and 6. Repetitive FISH patterns obtained with (GC)15 probe marked the pericentromeric region of the first chromosome pair. All probes showed accumulation in the microchromosomes. The chromosomal formula found in E. boulengeri has been considered the ancestral karyotype for pleurodont Iguania. The genus Enyalius is characterized by two distinctive chromosomal groups; one with highly conserved karyotypes, whereas the other is karyotypically diverse. Our molecular cytogenetics data are promising and will increase knowledge about the genus Enyalius chromosome evolution.