Abstract Genome changes, evidenced through karyotype or nuclear genome size data, can result in reproductive isolation, diversification and speciation. The aim of this study was to understand how changes in the karyotype such as chromosome number and nuclear genome size accompanied the evolution of neotropical stingless bees, and to discuss these data in a phylogenetic context focusing on the karyotype evolution of this clade. We sampled 38 species representing the three Neotropical Meliponini groups; 35 for karyotype analyses and 16 for 1C value measurement. The chromosome number varied from 2 n = 16 to 2 n = 34, with distinct karyotypic formulae and the presence of a few polymorphisms, such as B chromosomes in one species and arm size differences between homologous chromosomes in two species. The mean 1C value varied from 0.31 pg to 0.92 pg. We associated empirical data on chromosome number and mean 1C value to highlight the importance of Robertsonian fusion rearrangements, leading to a decrease in chromosome number during the Neotropical Meliponini evolution. These data also allowed us to infer the independent heterochromatin amplification in several genera. Although less frequent, Melipona species with 2 n = 22 represent evidence of Robertsonian fissions. We also pointed out the importance of chromosomal rearrangements that did not alter chromosome number, such as inversions and heterochromatin amplification.