Phlebotomine sand flies (Diptera: Psychodidae) are primary vectors of leishmaniasis. Greece and Turkey are both endemic for visceral and cutaneous leishmaniasis and are widely affected by the disease. Measures commonly applied for controlling sand flies rely on the use of insecticides, predominantly pyrethroids. A worldwide problem associated with the intensive use of insecticides is the development of resistance. Scarce information is available regarding the resistance status in sand fly populations. Sand flies were collected from Greece (Thessaloniki, Peloponnese, Chios island) and Turkey (Sanliurfa) and analyzed for the presence and frequency of target-site knockdown resistance mutations on the voltage-gated sodium channel (Vgsc) gene. Five sand fly species were included in the analysis: Phlebotomus perfiliewi Parrot, Phlebotomus neglectus Tonnoir, Phlebotomus simici Nitzulescu, Phlebotomus tobbi Adler and Theodor, and Phlebotomus papatasi Scopoli. Their Vgsc gene-domain II was analyzed for the presence of known pyrethroid resistance mutations. The mutation 1014F, associated with pyrethroid-resistant phenotypes, was detected in P. papatasi sand flies from Sanliurfa at an allele frequency of 48%. Homozygotes for the wild type allele 1014L (Leu/Leu) represented 36% of the population, while homozygotes for the resistant allele 1014F (Phe/Phe) and heterozygotes encompassing both alleles (Leu/Phe) each had a frequency of 32%. In all other sand fly species, only the wild type allele 1014L was detected. This is the first report for the detection of resistance mutations in the major leishmaniasis vector P. papatasi and is of major concern regarding leishmaniasis control.