Endosymbiotic Wolbachia bacteria have been previously shown to infect laboratory colonies of the human bed bug, Cimex lectularius L. (Heteroptera: Cimicidae), but little information exists regarding the extent of infection in natural populations. We assayed C. lectularius populations from five North American regions (California, Connecticut, Florida, New York, and Toronto, Canada) and one African region (Macha, Zambia) for Wolbachia infection by the polymerase chain reaction (PCR). Wolbachia infections were prevalent in all populations assayed (83–100%). There were no significant differences in infection frequency between geographic regions, between sexes, or between life stages (adult versus nymph). The potential utility of Wolbachia for alternative bed bug control strategies is discussed.