We performed a comparative analysis of the G- and C-banded karyotypes of seven species of didelphid marsupials, representing the three diploid numbers (2n = 14, 18 and 22) known to occur in this family. In addition to a great similarity among karyotypes with the same diploid numbers, we also identified homeologies for all autosomal arms comprising the three karyotypes. Robertsonian rearrangements, pericentric inversions and heterochromatin variation account for the differences among the karyotypes. Interspecific variation in the size of the sex chromosomes is due to differences in heterochromatic content. In-situ hybridization with total genomic DNA revealed considerable conservation of the euchromatic portions of the three karyotypes and indicated divergence of repetitive DNA sequences in autosomal heterochromatin.