Knowledge of the host-feeding pattern of blood-sucking insects helps to understand the epidemiology of a vector-born disease. A set of primers was used to selectively amplify segment of vertebrates' prepronociceptin gene from abdomens of engorged sand flies. Vertebrate DNA was successfully amplified in 65% of blood-fed phlebotomines assayed. Direct sequencing and comparison of resultant sequences with sequences in GenBank, using Basic Local Alignment Search Tool, led to the specific identification of the host in 100% of the cases. In total, 249 blood-fed females belonging to five different sand flies species were captured thanks to Centers for Disease Control and Prevention light traps and sticky papers in different areas of Tunisia between 2007 and 2009. Bloodmeal origin was determined for 146 blood-fed midges: Phlebotomus sergenti Parrot sampled fed only on Ovis aries and Equus caballus, while bloodmeal origin of P. perniciosus Newstead, P. longicuspis Nitzulescu, and P. papatasi (Scopoli) was diversified. We found that midges were fed mainly on Homos sapiens (n = 37; 22.69%), Bos taunts (n = 11; 6.74%), Mus musculus (n = 2; 1.22%), Capra hircus (n = 4; 2.45%), Camelus dromedarius (n = 3; 1.84%), Ovis aries (n = 98; 60.12%), Equus caballus (n = 3; 1.84%), Felis catus (n = 1; 0.6%), Oryctolagus cuniculus (n = 3; 1.84%), and Rattus norvegicus (n = 1; 0.6%). In this study, interestingly, we found for the first time that Mus musculus DNA was found in one female of S. minuta (Rondani) specie and question about its possible vectorial role is opened.