Tetranychus turkestani Ugarov and Nikolskii and Tetranychus urticae Koch RF (red form) (Acari: Tetranychidae) are closely related species. Previously, the two species were found in separate agricultural habitats in Israel. Here, additional collections were undertaken and mixed populations of the two species were found. Manipulation experiments were conducted in order to test whether sexual interactions occur when T. turkestani and T. urticae RF share the same host. Interspecific crosses showed that the two species are capable of producing viable F(1) females, but that these females are sterile as their F(2) eggs failed to hatch. This indicates a post-zygotic reproductive barrier, supporting the current placement of T. turkestani as a separate taxon. Mating behavior parameters revealed that males of both species courted virgin conspecific and heterospecific females at the same rate and readily tried to copulate with them. Female mate recognition seemed to be more reliable in T. turkestani than in T. urticae RF as the number of copulations was significantly higher and their duration significantly shorter in the T. turkestani interspecific (T. turkestani female x T. urticae RF male) as compared to the intraspecific crosses, a phenomenon not observed in T. urticae RF. In mixed cultures, a significant reduction in female production was observed for T. urticae RF but not for T. turkestani, suggesting an asymmetric reproductive interference effect in favor of T. turkestani. The long term outcome of this effect is yet to be determined since additional reproductive factors such as oviposition rate and progeny survival to adulthood may reduce the probability of demographic displacement of one species by the other in overlapping niches.