Tropidurus is a widespread genus of lizards in Brazilian biomes, particularly common in the state of Bahia, northeastern Brazil. However, genetic studies of populations and species of this group are still scarce. We used chromosomal and DNA barcoding analyses to examine 12 populations of five species of Tropidurus from semiarid (caatinga) and Atlantic forest portions of Bahia. All populations shared a karyotype with 2n = 36 divided into 12 metacentric and submetacentric chromosomes and 24 microchromosomes. The microchromosomes were acrocentric in most of species, but metacentric microchromosomes were observed in Tropidurus semitaeniatus, determining variation in fundamental number of arms from 48 to 52. One population of Tropidurus hispidus presented a supernumerary chromosome. Pericentromeric heterochromatin was detected in most microchromosomes and two pairs of macrochromosomes presented interstitial and pericentromeric C-bands. The nucleolar organizer regions (NORs) were single in Tropidurus erytrocephalus, T. semitaeniatus, and Tropidurus torquatus, and multiple in Tropidurus cocorobensis and T. hispidus. This is the first report of multiple NORs in both species. These results suggest a conserved pattern of karyotype macrostructure in Tropidurus, whereas microstructural features have undergone a more dynamic evolution, including non-Robertsonian rearrangements. DNA barcoding supported a clear distinction between analyzed species, except for the cryptic diversity indicated in the T. hispidus and T. torquatus cluster, in agreement with current taxonomic uncertainties. The diversity of this group of lizards is discussed based on their divergence and distribution patterns.