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.