Copulatory plugs serve as mating barriers in many animal species. We collected plugs and vaginal swabs from female banner-tailed kangaroo rats Dipodomys spectabilis in a wild population with all males individually genotyped, and used them as a source of DNA. Copulatory plugs solidly filled the reproductive tract, including the entrances to the uterine horns. Contrary to the popular hypothesis that plugs prevent females from remating, these plugs surprisingly contained DNA from up to three males. Alleles contributed by males were more numerous in internal sections of the plugs. Our results confirm that D. spectabilis females mate with multiple males and suggest that they avoid mating with close relatives. The apparent underrepresentation of DNA from related males implies precopulatory sexual selection, but postcopulatory mechanisms may also be at work. For example, our data are consistent with the possibility that pressure generated during plug coagulation promotes movement of sperm beyond cervical barriers. In controlled experimental settings, DNA from plugs will provide a powerful tool for distinguishing pre- and postcopulatory sexual selection.