We developed and tested a protocol for determining the sex of individual pinnipeds using the sex-chromosome-specific genes ZFX and ZFY. We screened a total of 368 seals (168 crabeater, Lobodon carcinophaga; 159 Weddell, Leptonychotes weddellii; and 41 Ross, Ommatophoca rossii) of known or unknown sex and compared the molecular sex to the sex assigned at the time of biopsy sample collection in the Ross and Amundsen seas, Antarctica. We also screened 6 captive northern elephant seals (Mirounga angustirostris) and 2 captive California sea lions (Zalophus californianus) of known sex. The assigned sex and genetic sex agreed for virtually all seals. Indeed, discrepancies ranged from 0.0% to 6.7% among species. It is not clear, however, if the few mis-assignments of sex occurred in situ or in the laboratory. The assigned morphological and molecular sex might both be correct with the discrepancies owing perhaps to developmental effects of environmental pollution. A subset of individuals sequenced at both loci revealed no intraspecific sequence variation. There was, however, sequence variation among species at both loci, which allowed them to be uniquely identified with as few as 2 and as many as 31 nucleotides.