Studies on conflict and cooperation in insect mating behavior usually assay the relative costs and benefits of mating in unreplicated experimental designs. We assayed multiple populations for such potential costs and benefits in Musca domestica L. under different nutritional environments. We used four populations, each derived from the same field site, ranging from six to 46 generations in the laboratory. Under benign environmental conditions (i.e., ad libitum food and water), mated females lived up to 13% longer than virgins, but this difference was not significant. Under extreme nutritional stress (i.e., no food or water), mated females lived significantly longer, but in only one of the populations tested. Under more moderate stress (i.e., water alone), mated females of one population died significantly earlier. Thus, aspects of both cooperation and conflict were found, but the effects were not repeatable across populations or nutritional environments. Overall, older females were more susceptible to stress. Moreover, the populations were significantly different in longevity (1.5-fold difference) and starvation resistance (2.2-fold difference). The youngest population lived longer, was more resistant to nutritional stress, and was the only line to exhibit significant costs of mating. Thus, the reduced selection pressures of the laboratory could have allowed the erosion of ejaculate toxicity, longevity, and starvation resistance. However, the simplest explanation is that replicate samples from a single geographic source population show appreciable among-line variation in fitness parameters and the consequences of mating. In conclusion, sexual selection studies on the costs and benefits of mating should consider environmental conditions, evolutionary history, and, perhaps most importantly repeatability.