The degree of reproductive isolation between the B and Q biotypes of the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) is currently not clear. Laboratory experiments have shown that the two biotypes are capable of producing viable F1 hybrids but that these females are sterile as their F2 generation failed to develop, indicating, most likely, a post-zygotic reproductive barrier. Here, we confirm, by molecular and ecological tools, that the B and Q biotypes of Israel are genetically isolated and provide two independent lines of evidence that support the existence of a pre-zygotic reproductive barrier between them. Firstly, monitoring of mating behaviors in homogeneous and heterogeneous couples indicated no copulation events in heterogeneous couples compared to ∼50% in homogeneous B and Q couples. Secondly, we could not detect the presence of sperm in the spermathecae of females from heterogeneous couples, compared to 50% detection in intra-B biotype crosses and 15% detection in intra-Q biotype crosses. The existence of pre-zygotic reproductive barriers in Israeli B and Q colonies may indicate a reinforcement process in which mating discrimination is strengthened between sympatric taxa that were formerly allopatric, to avoid maladaptive hybridization. As the two biotypes continued to perform all courtship stages prior to copulation, we also conducted mixed cultures experiments in order to test the reproductive consequences of inter-biotype courtship attempts. In mixed cultures, a significant reduction in female fecundity was observed for the Q biotype but not for the B biotype, suggesting an asymmetric reproductive interference effect in favour of the B biotype. The long-term outcome of this effect is yet to be determined since additional environmental forces may reduce the probability of demographic displacement of one biotype by the other in overlapping niches.