Berberis vulgaris (the barberry) as a well-known species in the family Berberidaceae is cultivated in many countries in the world. Iran with 11000 ha and more than 10000 tons of dried fruit per year, is the biggest producer of barberry in the world. In spring and summer 2017, severe dieback and decline of barberry trees were noticed in some old orchards in Abadeh (Fars province). In the closer and more accurate observations, typical branch cankers were frequently observed on some symptomatic trees. Various shapes of wood necrosis were found in cross section of branches of affected trees. Similar external and internal disease symptoms were also observed on barberry trees in different regions of Southern Khorasan province. Therefore, the aim of the present study was to determine the main fungal trunk pathogens associated with barberry trees showing disease symptoms in these regions of Iran. During each sampling, one to three branches (5–10 cm in diameter) were collected from affected trees and placed in individual plastic bags. Samples were taken to the laboratory for more examination of the internal wood disease symptoms and fungal isolation. Four to five cm-long tissue pieces were removed from each sample and the outer bark of them was cut away with a scalpel. From each piece 10-15 sections (4x4 mm) from the margin between healthy and necrotic wood tissues were aseptically removed. Small sections were sterilized by immersion in a 1.5 % sodium hypochlorite solution for two minutes, they were washed twice with sterile distilled water, and placed in Petri dishes (90 mm in diameters) containing potato dextrose agar (PDA) medium. Petri dishes were incubated at 25 °C in the dark for one to two weeks. All fungal colonies growing from the wood sections were isolated and sub-cultured on fresh media. All isolates were observed with an optical microscope and identified and grouped by the main macro and micro-morphological analyses. Morphological identifications of the representative isolates were confirmed by sequence analysis of the internal transcribed spacer (ITS) nrDNA region and a partial β-tubulin (BT) and translation elongation factor 1α (EF-1α) genes. A number of fungi, belonging to six genera including Phaeoacremonium, Neoscytalidium, Lasiodiplodia, Biscogniauxia, Paecilomyces, Fusarium and Alternaria were obtained from barberry trees. Of these, three genera were identified to the species level. These include Phaeoacremonium alvesii, Phaeoacremonium parasiticum, Neoscytalidium hyalinum and Lasiodiplodia theobromae. This is the first report of these species on B. vulgaris in the world.