The banding patterns of mitotic chromosome from three cottontail species, Sylvilagus aquaticus (2n = 38), S. floridanus (2n = 42), and S. transitionalis (2n = 46), are presented and compared with those of the proposed leporid ancestral karyotype, the latter being reflected in a species of hare, Lepus saxatilis (2n = 48). The differences in the diploid numbers of the cottontail species are primarily due to the fixation of Robertsonian fusions which have occurred during the evolution of their respective genomes. Of the eight fusion products identified, seven were species specific, while one, which is thought to reflect their more recent common ancestory, is shared by both S. aquaticus and S. floridanus. Other karyotypic differences include interspecific variation in the amount and distribution of constitutive heterochromatin, as well as the presence of two autosomal pairs in S. transitionalis which do not have apparent banding homologies either with the chromosomes of other cottontails or with those of the Lepus genome. The tendency for certain chromosomes in the ancestral karyotype to show a predisposition to undergo fusion events within the Leporidae is discussed.