Maternally inherited endosymbiotic bacteria Wolbachia pipientis have been shown to have wide-ranging effects on the reproduction of their hosts. This study presents the first survey and characterization of Wolbachia pipientis that have infected spiders collected from Wuhan, Hubei Province, China. First, we used universal primers of the wsp gene (Wolbachia Surface Protein, WSP) to examine the infection of Wolbachia in spiders. We found that, out of 31 spider species, 7 species were infected with Wolbachia. Then we used the specific primers of the A and B Wolbachia supergroups for the wsp gene to determine if there are super-infections in these infected spiders. Specimens of Nephila clavata were infected with strains of both A and B Wolbachia, while the others were infected with either strain A or B. Lastly, we aligned the sequences obtained with published ones to establish the phylogenetic relationships among Wolbachia found in spiders. The Wolbachia in Larinia argiopiformis Bösenberg & Strand 1906, Eriovixia cavaleriei (Schenkel 1963), Araneus ventricosus (L. Koch 1978), and Pholcus crypticolens Bösenberg & Strand 1906 belong to the A supergroup and the other three species, Nephila clavata (L. Koch 1878), Oxyopes sertatus L. Koch 1877 and Coleosoma octomaculatum Bösenberg & Strand 1906 belong to the B supergroup.