The karyotypes of 23 specimens belonging to 16 species from nine genera of Brazilian marsupials (family Didelphidae) were studied. The animals were collected in eight localities of Cerrado or Atlantic Forest biomes in the states of Goiás, Tocantins and São Paulo. The karyotypes were analyzed after conventional Giemsa staining and silver staining of the nucleolus organizer regions (Ag-NORs). New karyotypic data were obtained for Gracilinanus microtarsus (2n = 14, FN = 24), Marmosops paulensis (2n = 14, FN = 24) , Micoreus paraguayanus (2n = 14, FN = 20) and Monodelphis rubida (2n = 18, FN = 32) and are discussed in detail. The karyotypes of G. microtarsus , M. paulensis and M. paraguayanus include three large pairs of submetacentrics (pairs 1, 2 and 3) and a medium-sized metacentric or submetacentric pair 4. Pairs 5 and 6 are small submetacentrics in G. microtarsus and M. paulensis and acrocentrics in M. paraguayanus . M. paulensis presented a single Ag-NOR in pair 6 (6p6p), while M. paraguayanus exhibited multiple Ag-NORs in pairs 5 and 6 (5pq5pq6p6p). There was variation in size and morphology of the sex chromosomes among these species. Monodelphis rubida presented a karyotype with 2n = 18 and FN = 32 composed of a large submetacentric pair 1, a medium-sized metacentric pair 2 and six pairs of submetacentrics (pairs 3 through 8). The X was a small acrocentric and the Y was dot-like. A single Ag-NOR bearing pair (5p5p) characterized M. rubida. Relevant karyotypic information was obtained for 19 specimens belonging to 12 species collected in areas sampled for the first time [ Caluromys lanatus and C. philander (2n = 14, FN = 20), Gracilinanus emiliae (2n = 14, FN = 24), Marmosa murina , Metachirus nudicaudatus and Micoureus demerarae (2n = 14, FN = 20), Monodelphis americana (2n = 18, FN = 32) and M. domestica (2n = 18, FN = 20), and Didelphis marsupialis, Philander frenata, P. opossum and P. sp (2n = 22, FN = 20)]. Although the karyotypes were relatively conserved with respect to the morphology of the autosomes among species with the same diploid number, some differences regarding FN, sex chromosomes morphology and Ag-NORs patterns were detected.