Guadeloupe islands are threatened by several mosquito-borne viruses such as Dengue, Chikungunya, Zika and West Nile virus. It appears essential to look for alternative mosquito control methods such as the incompatible insect technique (ITT) aiming at sterilizing wild females by inundative releases of incompatible males. Before considering the implementation of such a strategy, the characterization of genetic diversity of the endocellular bacterium Wolbachia regarding the local mosquito populations is a critical issue. Here, for the first time, we describe the prevalence and diversity of Wolbachia in natural populations of three mosquito species from Guadeloupe: Aedes aegypti, Aedes taeniorhynchus and Culex quinquefasciatus. The detection of Wolbachia in natural Ae. aegypti, Ae. taeniorhynchus and Cx. quinquefasciatus populations was conducted by studying Wolbachia 16S ribosomal RNA gene using a TaqMan quantitative real-time PCR and results were confirmed by conventional PCR and sequencing. In addition, molecular typing of wPip strains in Cx. quinquefasciatus was done by PCR-RFLP. We did not find Wolbachia infection in any of Ae. aegypti and Ae. taeniorhynchus studied populations. Natural Wolbachia infection was detected in Cx. quinquefasciatus with prevalence varying from 79.2% to 95.8%. In addition, no polymorphism was found between the Wolbachia strains infecting Cx. quinquefasciatus specimens, all carrying an infection from the same Wolbachia genetic wPip-I group. These results pave the way for the evaluation of the feasibility of IIT programs to fight against these medically-important mosquito species in Guadeloupe.