We sequenced 540 nucleotides of the last exon in the ZFY/ZFX gene in two males and two females for eight cetacean species; four odontocetes (toothed whales) and four mysticetes (baleen whales). Based upon the obtained nucleotide sequences, we designed two sets of oligonucleotide primers for specific amplification of the ZFX and the ZFY sequence in odontocetes and mysticetes, respectively. Each primer set consisted of three oligonucleotides; one forward-orientated primer, which anneals to the ZFY as well as the ZFX sequence, and two reverse-orientated primers that anneal to either the ZFX or the ZFY sequence. The resulting two amplification products (specific for the ZFY and ZFX sequences) can be distinguished by gel-electrophoresis through 2% NuSieve™. The accuracy of the technique was tested by determination of gender in 214 individuals of known sex. Finally we applied the technique to determine the sex of 3570 cetacean specimens; 2284 humpback whales, 315 fin whales, 37 blue whales, 7 minke whales, as well as 592 belugas, 335 narwhals and 25 harbour porpoises.