Wolbachia are a group of maternally inherited bacteria harbored by a variety of arthropods and can ma- nipulate the reproductivity of their hosts. Eighteen populations of whiteflies were collected from Hebei, Xinjiang, Beijing, Shandong, Zhejiang, Guangxi, Hainan, Guangzhou and Fujian provinces, China. These whiteflies were molecularly identified using internal transcribed spacer 1 ITS1 rDNA sequences. They were then detected for Wolbachia infection using Wolbachia-specific primers designed based on 16S rDNA and wsp gene sequences. The results showed that almost all populations were detected positive for Wolbachia infection. Whiteflies of B / Q group mainly carried Wolbachia belonging to supergroup A, while non-B / Q whiteflies were commonly detected for Wolbachia superinfection. This study indicated that the infection rate of Wolbachia in natural populations of B. tabaci might be much higher than expected, and the threshold of detection methods may be one of the key factors influencing detection of Wolbachia infection.