In this work, we studied chromosome morphology, Ag-NOR, and C-banding patterns in the Neotropical leiuperid frogs that compose the Physalaemus henselii species group: Physalaemus fernandezae, Physalaemus henselii, and Physalaemus riograndensis. The chromosome diploid complement in all species was 2n  =  22 and consisted of seven large to medium and four small chromosome pairs. Chromosome fundamental number (FN) varied, because pair 11 was metacentric in P. henselii and P. riograndensis (FN  =  44) but telocentric in P. fernandezae (FN  =  42). Each species presented a single pair of Ag-NORs, which are located in the secondary constriction of pair 11 in the case of P. fernandezae, in pair 8 in P. riograndensis (as in many other Physalaemus species), and in pair 5 in P. henselii, which is a unique character state among leiuperids. There were no substantial interspecific differences in C-banding pattern, and the heterochromatic bands were mainly located in the centromeric regions of all chromosomes. Noncentromeric C-bands adjacent to NORs were detected in P. riograndensis and P. fernandezae, as already reported for other species of this genus. The karyotype of P. fernandezae was quite similar to that of species in the P. signifer group and of the leiuperid Eupemphix nattereri. Neither karyotypic data nor adult and larval morphology support the P. henselii species group as currently defined.