The DNA probe Y97 was derived from a repeat sequence in the human Y centromere, a region which must be present in a mitotically functional Y chromosome. We have demonstrated that Y97, which detects a Y-specific 5.5-kb EcoRI fragment by Southern analysis, is very useful for the molecular detection of small amounts of Y-derived material and represents a significant improvement over previous tests for molecular diagnosis of sex. The male-female difference in hybridization was unequivocal even when only 25 ng of total DNA was used per lane. Furthermore, in mixing experiments the 5.5-kb EcoRI fragment was detectable even when only 5% of the total DNA was male. By increasing hybridization stringency, we have developed a rapid, sensitive, and accurate method to detect Y chromosomal DNA in unrestricted samples.