Using a polymerase chain reaction (PCR) assay with primers designed for the Wolbachia 16S rRNA and outer surface protein (wsp) gene, we screened 11 laboratory colonies and 4 field samples of 6 sand fly species for Wolbachia infection. Infection was only detected in 3 laboratory colonies of Phlebotomus papatasi Scopoli, originally collected in Israel, Egypt, and Saudi Arabia.