Pistacia vera L. is an important crop species in the Southeastern region of Turkey. Alternaria blight disease caused by Alternaria spp. has become epidemic for the last couple of years. Disease symptoms mainly develop on young fruits and infections continue until harvest. Repeated infections decrease fruit quantity and quality that in turn reduce the yield. Pistachio fruits with Alternaria blight symptoms were collected during 2010-2011 growing seasons. Infected fruits were sampled from Adiyaman, Gaziantep, Sanliurfa, Mardin, Kahramanmaras and Siirt provinces where large pistachio plantations are located. Sampling and pathogen isolations were started in April just after pollination and fruit set, and continued until October, which is the harvest time of pistachio in the Southeastern Anatolia region. Isolates obtained from tissues infected with Alternaria blight were characterized morphologically and analyzed with DNA sequences, the nuclear internal transcribed spacer region, ITS and allergen regions, ALT, for molecular confirmation. Twenty-three Alternaria spp. isolates were obtained and subjected to pathogenicity analysis on 'Ohadi' pistachios. All the Alternaria spp. isolates caused blight symptoms on mature pistachio fruits. Four isolates were found to be highly pathogenic and there was difference among isolates in virulence levels. One highly virulent isolate was used to test reactions of 'Uzun', 'Ohadi' and 'Siirt' cultivars under field conditions, and 'Uzun' was found to be the most tolerant cultivar followed by 'Siirt' and 'Ohadi'.