A karyotypic and autoradiographic study was undertaken in an effort to identify the Y chromosome in five subspecies of the deer mouse, Peromyscus maniculatus. Three cells from each of seven male animals were analyzed. Although three of the animals were from the same subspecies and two were from the same location, all seven animals had different karyotypes. The existence of large groups of similar chromosomes, more than one heteromorphic pair of chromosomes or cell-to-cell variation within an animal precluded karyotypic identification of a Y in four animals. However, in all seven animals a probable Y could be designated from autoradiography since one chromosome consistently completed DNA replication later than the rest of the complement. The Y as identified by karyotyping corresponded to the latest labeling chromosome in only two animals. Observations are also presented on the proposed X in males.