Although selection against hybridization is expected to generate prezygotic divergence in unimodal hybrid zones, such a pattern has been seldom described. This study aims to better understand how prezygotic mechanisms may evolve in such zones. We investigated prezygotic divergence between populations of two subspecies of mice (Mus musculus musculus and M. m. domesticus) located at the edges of their unimodal hybrid zone in Denmark, and we developed an original multiple-population choice-test design, which allows assessment of within and between subspecies variation. Our study demonstrates that a strong assortative preference characterises one of the two subspecies (musculus) and that urinary signals are involved in this subspecies recognition. Taking into account the specific genetic and geographical characteristics of the Danish hybrid zone, we discuss the influence of the above pattern on its fate and the mechanisms that could have favoured this prezygotic divergence, among which the role of recombined populations constituting the core of the zone.