Historical specimens of sea otters (Enhydra lutris) provide valuable information about the pre-bottleneck population. However, the sex of these specimens is often not known. We attempted to determine the sex of sea otters by means of polymerase chain reaction (PCR) amplification of the amelogenin and the zinc finger (ZFX/ZFY) regions. The PCR fragments of amelogenin amplified were same size regardless of the sex. On the other hand, we were able to distinguish the sex accurately by means of the sea otter-specific restriction enzyme (NlaIII) to digest a polymorphic site in the ZFX/ZFY region. This method was applicable to dental pulp from skull specimens with the use of novel inner primers (KH1/KH2). We suggest that with some modification, the ZFX/ZFY system can be applied to other species and to a variety of archaeological specimens.