Karyotypes of 127 individuals, representing 19 species of 10 genera of Brazilian marsupials (Didelphidae) were determined, with karyotypes of Gracilinanus emiliae and G. microtarsus, Marmosops incanus, Thylamys velutinus, and Philander frenata being described for the 1st time. The specimens were collected from 24 localities, in an area between 4°N (Surumú, Roraima) and 31°S (Mostardas, Rio Grande do Sul), in Brazil. Only 3 diploid numbers were found, 2n = 14 in G. emiliae, G. microtarsus, G. agilis, Marmosa murina, Marmosops parvidens, M. incanus, Micoureus demerarae, and T. velutinus; 2n = 18 in Monodelphis dimidiata, M. brevicaudata, M. kunsi, and M. domestica; and 2n = 22 in Chironectes minimus, Lutreolina crassicaudata, Didelphis albiventris, D. aurita, D. marsupialis, Philander opossum, and P. frenata.
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