The genus <i>Partamona</i> includes 33 species of stingless bees, of which 11 were studied cytogenetically. The main goal of this study was to propose a hypothesis about chromosomal evolution in <i>Partamona</i> by combining molecular and cytogenetic data. Cytogenetic analyses were performed on 3 <i>Partamona</i> species. In addition, the molecular phylogeny included mitochondrial sequences of 11 species. Although the diploid number was constant within the genus, 2n = 34, B chromosomes were reported in 7 species. Cytogenetic data showed karyotypic variations related to chromosome morphology and the amount and distribution of heterochromatin and repetitive DNA. The molecular phylogenetic reconstruction corroborated the monophyly of the genus and separated the 2 clades (A and B). This separation was also observed in the cytogenetic data, in which species within each clade shared most of the cytogenetic characteristics. Furthermore, our data suggested that the B chromosome in the genus <i>Partamona</i> likely originated from a common ancestor of the species that have it in clade B and, through interspecific hybridization, it appeared only in <i>Partamona rustica</i> from clade A. Based on the above, <i>Partamona</i> is an interesting genus for further investigations using molecular mapping of B chromosomes as well as for broadening phylogenetic data.