Barley leaf stripe disease is one of the most important barley diseases, caused by heterothallic ascomycete Pyrenophora graminea in Iran. To assess the sexual fertility status of isolates of fungi and determination of mating type idiomorphs, 121 isolates belonging to six populations were obtained from some barley cultivating areas of provinces West Azerbaijan (Miyndoab and Naghadeh), East Azerbaijan (Maragheh and Miyaneh), Ardebil (Borjlou) and Zanjan (Khorramdareh) were investigated during 2016. At first, the primer pairs of MAT1 (for ampification of MAT1-1) and primer pair of MAT2 (for ampification of MAT1-2) were designed. The frequencies of mating type idiomorphs were evaluated by amplifying the conserved α area in idiomorph MAT1-1 and HMG region in idiomorph MAT1-2, by using multiplex PCR. The primer pairs of MAT1 and MAT2 amplify 826 bp and 719 bp, respectively. Among 121 isolates, 56 isolates were examined by MAT1 primer pair and 65 isolates by MAT2 primer pair. Frequencies of two mating type idiomorphs in Khorramdareh (1) and Miandoab populations had an excess of the MAT1-1 and MAT1-2 idiomorphs, respectively. According to the results of the Chi-square test, Miyaneh, Ardebil (1), Ardabil (2) and Khoramdareh (2) populations did not show significantly difference in the 5% level other than the Miandoab and Khorramdareh (1) populations (P <0.05). Therefore, these four populations possess the highest potential for sexual reproduction than the two populations of Miyndoab and Khorramdareh (1). In addition, 50 selected isolates belong to all populations were evaluated for sexual compatibility by crossing on the Suchʼs Agar culture medium enriched with barley straw, seed and young leaves. After six months, pseudothecia (ascocarp) containin no asci and ascospores formed in media.