The taxonomic status and distribution of the morphologically similar members of the Phlebotomus major complex in Turkey are unclear. To examine the utility of traditional morphological characters and molecular markers, sand flies were sampled from 90 localities in eleven different provinces covering a wide geographical range throughout Turkey. The morphometric variability was analysed using multivariate analyses of twelve characters, while mitochondrial cytochrome b (Cyt b) and nuclear elongation factor 1 alpha (EF 1-alpha) genes were used for molecular discrimination. Three distinct monophyletic lineages were identified based on the phylogenetic analysis of the combined data set of mitochondrial and nuclear gene regions, which were also supported by parsimony haplotype network analysis and AMOVA of Cyt b. The first lineage is restricted to south eastern Turkey and represents the species Phlebotomus syriacus, the second is present mostly in the westernmost and the easternmost localities and represents P. neglectus, and the third member of this complex is distributed across the mid-northern and mid-southern regions. None of the studied morphological characters were found to be sufficient to discriminate between these three members of the P. major s.l. complex; however their presence sympatrically in several localities supports their status as species rather than inter-population variability. (C) 2013 Elsevier B.V. All rights reserved.