AbstractThe stingless bee Partamona helleri (Meliponini) is distributed across the Atlantic rainforest biome in Brazil. Cytogenetic studies on P. helleri have shown a conserved diploid number and the presence of several B chromosome types. Our goal was to investigate the intraspecific karyotypic variation among 21 colonies of P. helleri from 14 different localities across four Brazilian states, using classical and molecular cytogenetics, as well as image cytometry (ICM). All populations had 2n = 34 and up to 5B chromosomes, which showed inter- and intra-population variation in number and size. C-banding evidenced the centromeric/pericentromeric heterochromatin and the partially heterochromatic B chromosomes. GA(15) microsatellite hybridized in the euchromatic arm of the A chromosomes and did not mark the B chromosomes, whereas TTAGG(6) corresponded to the telomeric sequence of the A and B chromosomes. The 18S rDNA and CMA3+ sites varied among the colonies, indicating the karyotype variability of the species. The ICM demonstrated an increase in nuclear genome size regarding the presence of B chromosomes. We highlighted the importance of population studies in species with a wide range of distributions, and how the B chromosomes contribute to the intra- and inter-population variability of P. helleri.