Few methods are available for determining the sex of free-ranging individual whales, dolphins, and porpoises of species that are not obviously sexually dimorphic. We have developed a technique for sexing beluga whales (Delphinapterus leucas) by using a Y-chromosome-specific DNA restriction fragment. Genomic DNA was extracted from liver samples of 18 beluga whales (9 males, 9 females) sexed at dissection. DNA from males and females was digested with five restriction enzymes, electrophoresed, and transferred to membranes by Southern blotting. When probed with the labelled human Y-chromosome zinc finger protein gene probe pDP1007, male-specific bands and bands common to both sexes, but more intense in females than in males, were observed. The DNA digested with EcoRI provided the clearest sex-discriminating banding pattern. Even when DNA of various qualities digested with EcoRI was used, all the males showed a 3.4-kilobase (kb) band, presumably from the Y-chromosome, as well as a 2.1-kb band. Females showed the 2.1-kb band, but all lacked the 3.4-kb band. This 3.4-kb EcoRI male-specific band permits unambiguous sex determination, which will facilitate examination of sex-related differences in population structure and habitat use of belugas, which have important implications for management decisions.