A VpreB surrogate light (SL) chain was identified for the first time in a marsupial, the opossum Monodelphis domestica. Comparing the opossum VpreB to homologues from eutherian (placental mammals) and avian species supported the marsupial gene being VpreB3. VpreB3 is a protein that is not known to traffic to the cell surface as part of the pre-B cell receptor. Rather, VpreB3 associates with nascent immunoglobulin chains in the endoplasmic reticulum. Homologues of other known SL chains VpreB1, VpreB2, and λ5, which are found in eutherian mammals, were not found in the opossum genome, nor have they been identified in the genomes of nonmammals. VpreB3 likely evolved from earlier gene duplication, independent of that which generated VpreB1 and VpreB2 in eutherians. The apparent absence of VpreB1, VpreB2, and λ5 in marsupials suggests that an extracellular pre-B cell receptor containing SL chains, as it has been defined in humans and mice, may be unique to eutherian mammals. In contrast, the conservation of VpreB3 in marsupials and its presence in nonmammals is consistent with previous hypotheses that it is playing a more primordial role in B cell development.