When two species come into contact and interbreed, the production of unfit hybrids can limit or prevent gene flow between the populations, thus maintaining each species' separate identity. The genetic basis of this hybrid dysfunction has recently begun to be elucidated, particularly for hybrid sterility and inviability. Although these dysfunctions can certainly act as a barrier to gene flow, other post-zygotic barriers may also play an important role in isolating species from one another. This study examines the genetic basis of the more subtle mechanism of species isolation via a marked reduction in lifespan of interspecies hybrid offspring. We found that females with homozygous X chromosomes in an otherwise interspecies hybrid background displayed a significant reduction in lifespan; this effect is not due to genetic background and appears to arise from complex genetic interactions. Separately, there is an additional severe reduction in lifespan for attached-X females when they have mated with males of either parental species, which is partly due to interspecific genetic interactions, but primarily due to a female's increased sensitivity to mating when bearing a Y chromosome or the attached-X chromosome construct.