Many important pathogens of crops worldwide are members of section Alternaria within the genus Alternaria. Representative species in this section such as Alternaria alternata, Alternaria tenuissima, and Alternaria arborescens show high variability, intermediate characters and plasticity in morphological features, which makes species identification difficult. The aim of this study was to characterize Alternaria species associated with pistachio and wild relatives in Turkey using molecular phylogenetics. One hundred isolates of Alternaria spp. from pistachio and wild relatives from Turkey were investigated. In addition, standard morphological reference isolates and Alternaria blight pathogens of pistachio from USA were included. Sequence data from major allergen a1, ATPase, endopolygalacturanase, and anonymous regions OPA1.3 and SCAR2 were obtained. Gene trees were estimated based on maximum parsimony, maximum-likelihood, and bayesian inference methods. Species tree estimation was performed based on Yule speciation and strict molecular clock assumption. Among the collection of Alternaria spp. from Turkey, only one A. arborescens isolate and three isolates which were morphologically A. alternata/A. arborescens intermediate types, but, phylogenetically close to A. arborescens were discovered. While A. alternata and A. tenuissima formed one phylogenetic species, A. alternata/tenuissima were phylogenetically distinct from A. arborescens. Furthermore, a TaqI restriction site in the endopolygalacturanase gene was explored as a novel diagnosis for identification of A. alternata/tenuissima and A. arborescens. All these molecular phylogenetic approaches allow to distinguish morphologically similar Alternaria pathogens and molecular phylogenies of Alternaria pathogens from pistachio and wild relatives in Turkey are described for the first time.