Intersexual opossums (Monodelphis domestica) from a large captive colony are described. These are the first naturally existing New World (didelphoid) intersexual marsupials for which reproductive phenotype and sex chromosome constitution are reported. One animal was XX, two were XY, and two were XO; all had lower body weight than normal males or females and the overall appearance of females. They were first recognized as abnormal by the presence of a small flaccid, nonstalked scrotum, markedly smaller than the scrotum of a normal male but in an equivalent position cranial to the cloacal aperture. Each scrotum contained a core of fatty connective tissue, but none contained testicular tissue. Teat patterns, seen only after close shaving of the hair over the area of the teat field, varied within and between the various sex genotypes, with one XY and one XO having the paired rudiments typical of normal males. All individuals had gonads, with no transabdominal migration. In the XX intersex there were mature ovaries with Graffian follicles, but in the XY and XO intersexes there was gonadal dysgenesis. The urogenital tract of all was female in appearance but was immature except in the XX intersex. Development of the scrotum and of the teat primordia can be explained on the basis of regulatory gene influences on the X chromosome. Intersex incidence in the colony is probably much higher than that observed because of ascertainment bias.