AbstractThe stingless bee genus Trigona includes 32 species, exclusive to the New World, which are grouped into two clades (A and B) according to phylogenetic molecular data. Cytogenetic studies have been performed in only seven Trigona taxa, and molecular cytogenetic data are available for only one species. These studies have been important for the chromosomal characterization of the species; however, discussions focusing on the karyotype evolution of Trigona in a phylogenetic context are lacking. In this study, we characterized the karyotype, through classical and molecular cytogenetics, of five Trigona species: T. pallens and T. williana, from clade A, and T. hypogea, T. aff. fuscipennis, and T. truculenta, from clade B, in order to provide insights into the karyotype evolution in Trigona and investigate whether the analyzed cytogenetic markers may have a phylogenetic signal. All five Trigona species have 2n = 34 chromosomes. Variations in the karyotype formula were observed in T. truculenta and T. hypogea compared with the other three species. Although heterochromatin distribution was restricted to one of the arms in most of the chromosomes of the five species, C-banding experiments highlighted a lower degree of heterochromatin compaction in T. pallens and T. williana. The microsatellite (GA)15 was exclusively located in the euchromatic regions of the chromosomes in all five species. The number of pairs bearing rDNA genes varied among the species studied, and this cytogenetic trait seems to reflect the phylogeny, separating the species into two clades. This study showed interspecific variations to a greater or lesser degree among Trigona species, highlighting the intense chromosomal evolutionary dynamics in the genus.