The mouse-like marsupial Antechinus agilis is common to south-eastern Australia with breeding and life-history traits being highly synchronous. Mating activity is confined to a 10- to 15-day period in August, at the end of which all males die as a result of a stress-induced suppression of the immune system. Ovulation occurs at this time and females rely on stored sperm from specialized crypts in the lower isthmus of the oviduct for successful fertilization. Here we report a high incidence of mixed paternity litters, which can be attributed to sperm from multiple males being stored in the isthmic crypts. Data from this study also suggest a possible second male siring advantage in controlled ex situ sperm competition mating trials, irrespective of the delay between the two males being given mating access to the female or of the mating time relative to ovulation. In determining paternity through DNA profiling, population genetic data were obtained that showed significant differences in the genetic heterozygosity between unrelated adults, half siblings and full siblings. We suggest that the isthmic crypts, in addition to storing viable sperm, are capable of releasing a mix of sperm that increases the likelihood of mixed paternity litters. This allows all mating males the opportunity to sire young, increases female reproductive fitness and overall maintains high levels of population genetic heterozygosity in the face of total male mortality annually.