Marsupial karyotypes have shown extensive conservation even between distantly related groups with a high diversity of life forms and reproductive biology. Banding analysis has been the main test for assessing their homologies and chromosome rearrangements. More recently, cross-species reciprocal chromosome painting has been developed and applied to several mammalian species and has shown homologies and rearrangements not revealed by banding analysis. Karyotype relationships between four marsupial species, Sminthopsis crassicaudata, Potorous tridactylus, Trichosurus vulpecula and Macropus eugenii, which are from different families in two orders, were investigated and presented in the form of comparative chromosome maps. These show that only a limited number of chromosomal rearrangements have occurred during their evolution. A karyotype phylogeny of the four marsupials was derived from these maps. A comparison between published gene location and the comparative chromosome maps for these species is presented and inconsistencies with previous gene mapping data indicated.