The pistachio nut tree (Pistacia vera) is one of the most important agricultural products in Iran. Crown and root rot which caused by fungi is the most important diseases of this plant in Iran. So far, several fungi have been identified as the cause of this disease. The causal agent of this disease is unknown in Roshtkhar county in Razavi Khorasan Province with more than 8000 hectares of pistachio garden. This research was carried out with the aim of isolating and identifying the fungi associated with root and crown rot of pistachio in Roshtkhar county. A total of 70 pistachio gardens with crown and root rot disease were selected and sampled during different seasons in the period 2015 to 2017. Sampling was done from pistachio trees with crown and root rot symptoms and soil from the diseased plant rhizosphere. Fungal isolates were recovered from surface disinfected tissues and soil samples by using the general and specific media culture. Identification of the isolates was done based on the colonial and morphological characteristic. Genomic DNA was extracted by the CTAB method and replicated with ITS1 and ITS4 primers. 42 fungal isolates were recovered from infected pistachio tissues. Based on the morphological and the sequences of ITS region, fungal isolates of this research belonged to Alternaria alternata, Ulocladium atrum, Rhizoctonia solani, Clonostachys rosea, Truncatella angustata, Fusarium equiseti, Aspergillus niger, Penicillium marneffei and F. solani species. This is the first report of these fungal species from the crowns and roots rot of pistachio from Roshtkhar county.