Wolbachia pipientis (Rickettsiales: Rickettsiaceae) protects mosquitoes from infections with arboviruses and parasites. However, the effect of its co-infection on vector competence for Dirofilaria immitis (Spirurida: Onchocercidae) in the wild has not been investigated. This study aimed to screen vectors of D. immitis for wPip, to characterize these, and to investigate a possible association between the occurrence of W. pipientis and that of the nematode. The presence of W. pipientis was assessed in the five mosquito potential vectors of D. immitis in Portugal. Polymerase chain reaction (PCR) products were sequenced, and wPip haplotypes were determined by PCR–restricted fragment length polymorphism (RFLP). Results showed that wPip was detected in 61.5% of Culex pipiens (Diptera: Culicidae) pools and 6.3% of Culex theileri pools. wPip 16s rRNA sequences found in Cx. theileri exactly match those from Cx. pipiens, confirming a mosquito origin, rather than a nematode origin, as some specimens were infected with D. immitis. Only wPip haplotype I was found. No association was found between the presence of wPip and D. immitis in mosquitoes and hence a role for this endosymbiont in influencing vectorial competence is yet to be identified. This study contributes to understanding of wPip distribution in mosquito populations and, to the best of the authors' knowledge, is the first report of natural infections by wPip in Cx. theileri.