Stigmaeopsis celarius Banks (hereafter Sc) is a spider mite living and feeding on the leaves of various bamboo species such as Moso bamboo [Phyllostachys edulis (=P. pubescens)] and Pleioblastus spp. (Poaceae). A previous phylogenetic study revealed a cryptic, phylogenetic sister species to Sc (hereafter Ss). Although its life type appears to be similar to that of Sc, individuals of Ss make much smaller nests compared with Sc, and the nests have been found mostly on Nezasa bamboo (Pleioblastus argenteostriatus). To investigate whether Sc and Ss are reproductively isolated, we explored their populations in southwestern Japan, and crossed them to examine mating behaviors and fertilization success. Field surveys revealed that the nests of these two species occur on the same leaves and, thus, the individuals of these species may make frequent contact. Reciprocal crosses suggested that the two species are reproductively isolated. Though Sc males have tried to mate with Ss females, copulation seldom occurred because of their long opisthosoma (hind body), which prevented the insertion of the aedeagus into the genitalia of Ss females. In contrast, most Ss males ignored Sc females, and eggs were not fertilized even in the few cases where copulation appeared to occur. These results suggest that strong selection pressure is imposed on body length to prevent interspecific hybridization in the contact area of these species.