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Abstract The role of hybridization as a formative process in evolution has received much attention in the past few decades. A particularly fascinating outcome of hybrid speciation is the formation of asexual hybrid species. The Amazon molly (Poecilia formosa) is such a hybrid and originated from aP. mexicanamother and aP. latipinnafather. Consequently, a heterospecific mating must have occurred leading to the Amazon molly, indicating a breakdown of any potential prezygotic isolation between parental species. Here we studied the female mate preferences of extantP. mexicanaandP. latipinnafrom several populations using standard binary choice tests with males of both sexual species that were matched for size.Poecilia mexicanaandP. latipinnacan be crossed in the lab, however, the offspring are not asexual, but sexual F₁s. In our study, we generated F₁s and tested their mating preferences with sexual males of bothP. mexicanaandP. latipinnaagainst F₁males. Overall, our results show that in extantP. mexicanaandP. latipinnano female preference for conspecific males was detectable. Consequently, heterospecific matings are possible and not hindered by any apparent behavioral prezygotic isolation. If female preferences in these species were comparable around the time the Amazon molly originated as a hybrid species ca. 100,000 years ago, matings leading to hybrids would be very likely. F₁females also have no discernable mating preferences for either sexual males or F₁males. Such lack of prezygotic behavioral isolation could potentially lead to F₂individuals, backcrosses, and introgression.