During 2012, soil samples from commercial pistachio orchards in three major production regions include Rafsanjan (Kerman Province, center of Iran), Damghan (Semnan Province, north-central Iran) and Feyz-Abad (Khorasan-e Razavi Province, northeastern Iran), were assayed on Dichloran Rose-Bengal Chloramphenicol agar (DRBC) and Aspergillus flavus-parasiticus agar media to quantify populations of Aspergillus species from the section Flavi. The mean propagule density of Aspergillus species from the Flavi section [log10 (CFU/g soil)] was higher in Feyz-Abad (3.06, 2.88–3.24), compared to Damghan (2.55, 2.44–2.65) and Rafsanjan (2.40, 2.26–2.54). A. flavus (69.7, 65.3 and 57.9%), A. parasiticus (19.6, 25.4, and 29.3%), and A. nomius (10.7, 9.3, and 12.8%) were the predominant species in the regions of Rafsanjan, Damghan, and Feyz-Abad, respectively. There were significant differences among sclerotia producing isolates of A. flavus in the sampling regions (p < 0.05). The percentage of sclerotium-producing isolates of A. flavus from Rafsanjan (14.5%) was much lower than Damghan (39.5%) and Feyz-Abad (41.4%). The A. flavus isolates from Damghan, Rafsanjan, and Feyz-Abad were toxigenic at 53.7%, 61.6%, and 60.4%, respectively. In Rafsanjan, aflatoxin B1 (AFB1), and AFB1 + AFB2 (aflatoxin B2) ranged from 274 to 553 ppb (393±17.11) and 394 to 3745 ppb, respectively, while AFB1, and AFB1 + AFB2 ranged from 257 to 392 ppb (285±13.18) and 415 to 1658 ppb, respectively, in Damghan. We found 16 and 20 vegetative compatibility groups (VCGs) for 41 and 37 nit mutant producing isolates of A. flavus from Rafsanjan and Damghan, respectively. From Damghan the VCG diversity for A. flavus isolates was greater (54%) than from Rafsanjan (39%). Because there were a few number of sclerotium-producing isolates of A. flavus, we did not determine the relationships between sclerotium production with VCGs and/or geographical distribution in the three pistachio production regions. This study was the first to determine the strain and VCG diversity of A. flavus soil isolates from Iranian pistachio orchards.