Studies were performed in an endemic area of Dirofilaria immitis in Spain to genetically characterize the potential mosquito vector species present by means of DNA sequencing and elucidate which of these species may be involved in the transmission. The rDNA ITS-2 sequences of two Culex pipiens haplotypes, H1 and H2, Aedes (Aedimorphus) vexans, Fredwardsius vittatus, Ochlerotatus (Ochlerotatus) caspius, Anopheles (Anopheles) atroparvus, and Anopheles (Anopheles) plumbeus were obtained. F. vittatus and An. plumbeus were detected for the first time. Results on abundance, presence, and activity suggest that the month of August is the period of higher transmission risk, with C. pipiens, Ae. vexans, and An. atroparvus present simultaneously. Population studies indicate that C. pipiens may be considered the most important potential vector, while Ae. vexans, An. atroparvus, and O. caspius being involved in transmission only sporadically. The absence of larval dirofilarial infection agrees with the very low prevalences known in endemic areas.