Plebeia (Hymenoptera: Apidae) is a poorly defined genus and its classification and systematics are controversial. Tools such as cytogenetics may contribute to clarify the relationships among the species. The aim of this study was to characterize the karyotypes of the species Plebeia lucii Moure, 2004 and Plebeia phrynostoma Moure, 2004. For this purpose conventional staining, C-banding and fluorochrome techniques were performed. The same chromosome number (2n = 34) was observed for both species. The karyotypic formula of P. lucii was 2K = 22 A^M 12A. A heteromorphic pair was observed with euchromatic and heterochromatic regions of different sizes on the 2 homologs. The presence of a secondary constriction was observed in this same pair. In P. phrynostoma the karyotypic formula was 2K = 18A^M 10A 6M and did not show polymorphisms or secondary constrictions. The DAPI fluorochrome marked portions of the heterochromatic arm and the regions close to the centromere in some chromosomes of both species. CMA₃ marked the heteromorphic pair in P. lucii and some points in other chromosomes, while it stained 2 pairs of chromosomes in P. phrynostoma. Despite the similarity in chromosome number, these species show variation both in morphology and in composition of chromatin which may reflect a phylogenetic position in different clades.