California produces 99.1% of pistachios grown in the United States, and diseases affecting pistachio rootstocks represent a constant challenge to the industry. Field surveys of fungi associated with pistachio rootstocks with symptoms of crown rot and stem canker in three central California counties followed by phylogenetic analyses of translation elongation factor 1-α and second largest subunit of RNA polymerase II gene fragments identified three Fusarium species (Fusarium equiseti, Fusarium oxysporum, and Fusarium proliferatum) and two Neocosmospora species (Neocosmospora falciformis and Neocosmospora solani). F. oxysporum and N. falciformis were the fungal species most frequently recovered from symptomatic pistachio trees. Inoculations of detached twigs of cultivar Kerman pistachio Pioneer Gold I and clonal University of California, Berkeley I (UCBI) rootstocks showed that all five species could colonize pistachio wood and cause vascular discolorations. Pathogenicity tests in potted pistachio trees completed Koch's postulates and confirmed that F. oxysporum, F. proliferatum, N. falciformis, and N. solani were capable of producing rot and discoloration in stems of clonal UCBI rootstocks, the most widely planted pistachio rootstock in California. To our knowledge, this study is the first to present insights into the biodiversity and biology of Fusarium and Neocosmospora species associated with pistachio trees in California.