The gray short-tailed opossum, Monodelphis domestica, is the world's most widely utilized marsupial model for biomedical research. Recent completion of the initial M. domestica genome assembly offers the first opportunity to examine genome-wide phenomena in a marsupial. Using in silico methods, we have mapped 124 conserved microRNAs (miRNAs) to 94 loci in the M. domestica genome. In addition, using RNA pooled from 5 tissues, we cloned 85 miRNAs. Seventy-two of these are conserved miRNAs that we had mapped in silico. The additional 13 are nonconserved candidate miRNAs in 11 loci. Nine of these 13 are also found in the wallaby (Macropus eugenii) genome. Two of the candidate miRNA clones, located on the X chromosome, are part of a cluster containing a total of 24 potential miRNAs spanning more than 100 kb.