Background: Candidatus Riesia pediculicola is the primary endosymbiont of human lice which is important to the louse survival. However, bioinformatic analysis of the Riesia genome revealed that its vitamin B1 pathways are not complete in Riesia. These findings suggest that a secondary louse endosymbiont may be present and responsible for the missing functions. . Wolbachia, an obligate intracellular alpha proteobacteria, is a candidate for this role, similar to secondary symbionts in other arthropod hosts including those for tsetse flies, aphids and many other insects with deficient diets. The purpose of this study is to determine the infection rate of Wolbachia in human head lice and if the rates differ from different geographic origins. Methods: DNA samples of human lice from the USA, Madagascar, and Russia are tested by PCR using Wsp81F and Wsp691r primers that can detect the single copy conserved Wsp gene of Wolbachia. PCR products are detected by 1% agarose gel electrophoresis and are sequenced to determine the genotype of Wolbachia. PCR assays targeting the Wolbachia FtsZ gene or 16S rRNA gene will be used if the Wsp PCR assay does not work well enough and to extend the phylogenetic characterization. Results: DNA of Wolbachia was amplified using Wsp gene primers from several whole DNA samples from spittle bugs to confirm the assay conditions. Its 611-bp wsp fragment was then cloned into a plasmid to produce a positive control needed for testing the louse samples. Males, females, and instars from Madagascar, Russia and Georgia, USA sites are being compared. Conclusions: This project will contribute to a better understanding of louse biology and of the symbiotic relationship between human lice and their bacterial flora. Confirming the widespread presence of Wolbachia in lice will provide an immediate impetus to new approaches in the research effort to control of lice and improve the treatment of pediculosis in humans.