In this study, 23S rDNA from Arsenophonus and wsp gene from Wolbachia were amplified by PCR method using specific primers to study their symbiosis in Chilo suppressalis from 20 locations of China. The results indicated that Arsenophonus and Wolbachia infection rates differed widely in C. suppressalis populations. The infection rates of Arsenophonus ranged from 5.0% to 50.0% in five geographical populations from Harbin, Huishui, Jilin, Nanyang, and Yangzhou. Wolbachia ranged from 25.0% to 40.0% in three geographical populations from Hanzhong, Nanning, and Yangzhou. Their symbiosis was not observed in C. suppressalis of other locations. The sequences of 23S rDNA gene which named csArs were exactly the same in the five populations. However, the sequences of wsp from the three strains of Wolbachia showed wChisup1 belonged to supergroup A, wChisup5 and wChisup6 belonged to supergroup B. The results showed the strains of Arsenophonus from C. suppressalis of the five different locations were identical, whereas the strains of Wolbachia were phylogenetically diverse. The phylogenetic analysis indicated that 23S rDNA and wsp sequences found in C. suppressalis were exactly the same with or very closely related to relevant sequences in other species.