From March through December 2010, the incidence of vaginal septa in our SPF breeding colony of BALB/cByJNarl mice was 14.2%. In general, septa obstructed half of the vaginal orifice. Here we sought to determine the effect of this defect by comparing the reproductive performance of affected (septate) mice with that of unaffected (nonseptate) mice. Our results showed that the rates of both copulatory plugs and pregnancy were significantly lower in septate mice than in nonseptate mice. Specifically, 23 of 45 bred septate female mice (51%) had vaginal plugs compared with 49 of 68 bred nonseptate females (72%). Only 12 septate female mice (27%) had successful pregnancies, compared with 37 nonseptate females (54%). Septate mice had a 1-logfold fewer intrauterine sperm after mating than did nonseptate mice. Three cases of dystocia were noted among septate mice whereas none occurred in nonseptate mice. Septate dams had a higher percentage of septate pups (15.5%) than did nonseptate dams (6.1%). Our findings indicate that vaginal septa affect the reproductive performance of laboratory mice and that such a defect should be considered as an exclusion criterion for the selection of future breeders in a mouse colony.