Study of genetic structure of Ommatissus lybicus de Bergevin revealed significant variation among the allopatric populations of this pest. High biological and behavioral variation suggest either that this is a highly variable pest species or a complex with a mix of cryptic species. The degree of reproductive isolation was tested by crossing experiments on four allopatric Iranian populations. These experiments were performed at 27 ± 2 ° C, 65 ± 5% RH, and a photoperiod of 14:10 (L: D) h. In addition, to examine the possible role of Wolbachia in accelerating speciation through reproductive isolation, a PCR-based Wolbachia detection and frequency assay was used. Intercross mating studies with four divergent populations demonstrated fecundity in all of the no-choice mating trials, while in some replicates of the interpopulation crossing treatments (Abu Musa × Tezerj and Bam × Tezerj), no adult insects were produced. These populations experienced repeated exposure to heavy pesticide applications, which may accelerate genetic divergence. Wolbachia was detected in specimens from two O. lybicus populations (Bushehr and Jahrom) with 100% infection rate. Results of no-choice mating trials supported reproductive isolation among O. lybicus populations, which may lead to rapid evolution and speciation. A comprehensive and ecofriendly management program with emphasis on judicious use of pesticides for O. lybicus management is recommended to reduce genetic diversification in this species.