Leishmaniasis is a vector-borne disease transmitted byphlebotominesand flies. Information about blood meal preferences in sand flies is essential to understand the epidemiology of the diseaseto adopt control measures.In previous studies, a polymerase chain reaction (PCR) of 359 bp fragment of the conserved gene cytochrome b (cyt b) and further sequencing were applied in the study of blood meal sources in sand flies collected in the area of a leishmaniasis outbreak insouthwest Madrid, Spain, providing significant information about blood meal preferences in the focus. In this work, aPCR-restriction fragment length polymorphism(RFLP)targeting afragment of 359 bpof vertebrate cytbgene was developed. Restriction endonucleasesHaeIII and HinfI generated specific patterns consistent with the blood meal sources found in sandflies. The protocol has been validatedwithtwenty sixengorged females collected in the field with CDC traps.Blood meals from nine vertebrateswere identified based on PCR-cytb and sequencing -human, dog, cat, horse, hare, rabbit, sheep, goat and chicken - and mixed blood meals (sheep/human;sheep/goat)
and successfully distinguished byPCR-RFLP. Therefore, thisapproachis anefficient and reliable alternative method to be applied in entomological surveys.