Reproductive character displacement is known to occur at the borders of a secondary contact zone between the two European subspecies of the house mouse (in Jutland, Denmark), where selection against hybridization occurs. This study assessed patterns of mate preference in naturally occurring hybrids of the two subspecies. Mate odour choice was investigated in male and female mice sampled across the hybrid zone. Odour samples comprised urine (from the opposite sex to the test animal) obtained from populations geographically distant from the hybrid zone. Urine is known to carry subspecies recognition signals. The behavioural results changed across the hybrid zone, and were analysed by a model of clinal variation. This behavioural cline was compared with the allozyme cline across the same hybrid zone. Males on both sides of the hybrid zone showed an assortative preference, which shifted significantly and abruptly approximately 10 km from the genetic centre of the hybrid zone on the Mus musculus musculus side. Directional preference was not detected in females, which could relate to variation in sexual receptivity. Our model indicates that the peculiar pattern of male preference could involve several genes and be characterized by mild to strong epistasis favouring the expression of M. m. domesticus-like preference over a large portion of the hybrid zone. This study may provide the first picture of the genetic determination of mate preference in a mammal.