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Abstract A preference for mating with conspecifics over heterospecifics is fundamental to the maintenance of species diversity in sexually reproducing organisms. This type of positive assortative preference results in sexual isolation, and a reduction in gene flow between species due to differences in mate choice. The proximate and ultimate causes of sexual isolation therefore constitute active areas of research in evolutionary biology. Sexual isolation is often stronger between closely related sympatric species as compared to allopatric species because of processes such as reinforcement. In addition, traditional theories of sexual selection suggest that because reproduction is more costly to females, they should be the choosier sex and play a more central role in sexual isolation. We conducted a comparative analysis of assortative mate preferences in males and females of sympatric and allopatric species pairs of darters (fish genusEtheostoma). We performed a meta‐analysis of 17 studies, encompassing 21 species, in which assortative preference was measured when fish were (in most cases) allowed only visual information. As expected, we found stronger preferences for conspecifics over heterospecifics across studies and species. However, we did not find an effect of sympatry or sex on the strength of preference for conspecifics, but rather remarkable variation across species. We offer several testable hypotheses to explain the variation we observed in the strength of assortative preference.