The contact zone between the Ancarano (ACR; 2n = 24) and Cittaducale (CD; 2n = 22) races of Mus musculus domesticus was studied. We used chromosomes and mitochondrial DNA (mtDNA) sequences of the control region as genetic markers to detect introgression between races. In total, 76 mice were trapped at 17 localities. Cytogenetic analysis was performed on 73 mice. A segment of the control region (468 bp) was sequenced in 41 specimens. The two races are distributed parapatrically and the contact zone was identified inside a village (Pizzoli). No mixed population was found in the study area. The contact zone does not correspond to any geographical or ecological barrier but is located in a zone of potentially high density of mice. The sequence analysis clearly demonstrates genetic differentiation between races (1.4% of sequence divergence). Hybridization is rare. Evidence of introgression was found in two individuals in the contact zone: one individual of the ACR race carries a metacentric belonging to the CD race, while another ACR individual carries a CD-like haplotype. In these ecological conditions, the observed distribution pattern and the very low level of hybridization suggest the presence of a premating mechanism of reproductive isolation.