Transfer and receipt of seminal fluid proteins crucially affect reproductive processes in animals. Evolution in these male ejaculatory proteins is explained with post-mating sexual selection, but we lack a good understanding of the evolution of female post-mating responses to these proteins. Some of these proteins are expected to mediate sexually antagonistic coevolution generating the expectation that females evolve resistance. One candidate in Drosophila melanogaster is the sex peptide (SP) which confers cost of mating in females. Here, we compared female SP-induced post-mating responses across three D. melanogaster wild type populations after mating with SP-lacking versus control males including fitness measures. Surprisingly, we did not find any evidence for SP-mediated fitness costs in any of the populations. However, female lifetime reproductive success and lifespan were differently affected by SP receipt indicating that female post-mating responses diverged among populations. Injection of synthetic SP into virgin females further supports these findings and suggests that females from different populations require different amounts of SP to effectively initiate post-mating responses. Molecular analyses of the sex peptide receptor suggest that genetic differences might explain the observed phenotypical divergence. We discuss the evolutionary processes that might have caused this divergence in female post-mating responses.