The phenomenon of B6-Y(DOM) sex reversal arises when certain variants of the Mus domesticus Y chromosome are crossed onto the genetic background of the C57BL/6J (B6) inbred mouse strain, which normally carries a Mus musculus-derived Y chromosome. While the sex reversal has been assumed to involve strain-specific variations in structure or expression of Sry, the actual cause has not been identified. Here we used in situ hybridization to study expression of Sry, and the critical downstream gene Sox9, in strains containing different chromosome combinations to investigate the cause of B6-Y(DOM) sex reversal. Our findings establish that a delay of expression of Sry(DOM) relative to Sry(B6) underlies B6-Y(DOM) sex reversal and provide the first molecular confirmation that Sry must act during a critical time window to appropriately activate Sox9 and effect male testis determination before the onset of the ovarian-determining pathway.