Wolbachia are obligate intracellular bacteria present in reproductive tissues of many arthropod species. It has been reported that few silverleafing populations of Bemisia tabaci were positive for Wolbachia, whereas non-silverleafing populations were more likely infected with Wolbachia and all that infect B. tabaci are Wolbachia belonging to supergroup B. However, current detection methods were shown to be not sensitive enough to uncover all infections. Herein, a protocol based on polymerase chain reaction-restriction fragment length polymorphism analysis of Wolbachia 16S ribosomal DNA is presented. A systematic survey for the prevalence of Wolbachia infection in natural populations of B. tabaci using this method revealed that (1) all populations of B. tabaci tested positive for Wolbachia and the overall infection rate reached 80.5% (293 positives in 364 tests); (2) both single infection and superinfection existed within individual whiteflies tested; and (3) silverleafing populations of B. tabaci most likely harbored A Wolbachia as single infection, whereas non-silverleafing populations tend to carry B Wolbachia as superinfection. It is clear that the Wolbachia infection pattern is closely related to the genetic races of B. tabaci, and the infection frequencies are apparently much higher than those described previously. This study shows that detection methods can significantly influence estimation of Wolbachia infection. It is supposed that Wolbachia may be acting as a biotic agent promoting rapid differentiation and speciation of B. tabaci. This is the most systematic survey of Wolbachia infection within B. tabaci.