An entomological survey was carried out in 2007 in two Pyrenean counties of Lleida province (northeastern Spain), where cases of autochthonous canine leishmaniasis have been recently reported. Phlebotomus ariasi and P. perniciosus, vectors of Leishmania infantum in the Mediterranean area, were captured. The aim of the present study was to compare these phlebotomine populations with others captured in known leishmaniasis foci in Europe. Populations of these species were studied by analysing the polymorphism of seven enzymatic systems (HK, PGI, PGM, MDH, 6PGD, FUM and ACO) and compared with other specimens from endemic regions of France, Italy, Malta, Portugal and Spain captured in other campaigns, and also with previously published results. Phlebotomus ariasi was more polymorphic than P. perniciosus. Only the ACO locus had diagnostic alleles, but some other alleles show high characteristic frequencies for each species. The neighbourjoining trees separated two population groups in both species. On the basis of the isoenzyme study results, sand fly populations of the Pyrenean region in Lleida province are closely related to those of other nearby leishmaniasis endemic regions in France and Spain.