Intracellular endosymbionts, Wolbachia spp., have been reported in many different orders of insects and in nematodes but not previously in fleas. This is the first conclusive report of Wolbachia spp. within members of the Siphonaptera. Using nested polymerase chain reaction (PCR) targeting of the 16S ribosomal RNA gene, we screened for Wolbachia spp. in fleas collected from 3 counties in Georgia and 1 in New York. The prevalence of Wolbachia spp. detected varied among the 6 different species screened: 21% in the cat flea Ctenocephalides felis (n = 604), 7% in the dog flea C. canis (n = 28), 25% in Polygenus gwyni (n = 8), 80% in Orchopeas howardi (n = 15), 94% in Pulex simulans (n = 255), and 24% in the sticktight flea Echidnophaga gallinacea (n = 101). Wolbachia spp. infection in fleas was confirmed by sequencing positive PCR products, comparing sequenced 16S ribosomal DNA (rDNA) with Wolbachia spp. sequences in GenBank using BLAST search, and subjecting sequence data to phylogenetic analysis. For further confirmation, 16S rDNA–positive samples were reamplified using the wsp gene.