The Red Queen hypothesis (RQH) is one of the most widely accepted hypotheses explaining the persistence of sexual reproduction despite its costs. It posits that sexual species, compared with asexuals, are more adept at countering parasites, because their per-generation recombination rate is higher. Despite theroretical support, current empirical studies have failed to provide unanimous support. Here, we suggest that future tests of the RQH should more thoroughly elucidate its underlying assumptions and potential alternative hypotheses. While the RQH predicts that negative frequency-dependent selection shapes host-parasite interactions, differences between sexuals and asexuals are potentially important. Key assumptions about asexual species and their sexual close relatives include (i) ecological and behavioral traits are similar, (ii) among-individual genetic diversity is greater in sexuals than in asexual, and (iii) within-individual genetic diversity is similar in asexuals and sexuals. We review current evidence for the RQH, highlight differences between asexual and sexual species and how those differences might translate into differential responses to parasite infections, and discuss how they can influence the results and interpretation of empirical studies. Considering differences between asexual and sexual species in future tests of the RQH will help to refine predictions and eliminate alternative hypotheses.