Hop stunt viroid (HSVd) infects a large number of woody plants such as grapevine, citrus, plum, peach, fig, mulberry, pear, pistachio, and almond. Kerman province is the most major pistachio‐growing region in Iran. During 2015–2016, pistachio cultivars were surveyed in the province to detect HSVd. A total of 106 asymptomatic and symptomatic leaf samples showing mosaic, yellowing, and chlorosis symptoms were collected from pistachio trees and assayed for the HSVd infection by reverse transcription polymerase chain reaction, and dot‐blot hybridization. Results showed that four pistachio cultivars (Momtaze, Kalleh‐Ghouchi, Ohadi, Ahmad-aghaei) are infected by HSVd, while no HSVd infection was detected in the Akbari cultivar. In this study, bioassay tests including the mechanical transmission of viroid to pistachio rootstocks were used to evaluate the biological characteristics of Iranian HSVd pistachio variants. The infectivity of one HSVd variant (Ker.Ana.P1) was further verified by mechanical inoculation. The viroid variant was transmitted to two different healthy pistachio rootstocks (Badami, University of California Berkeley I (UCB-1)) using infected sap by three inoculation methods i.e. slashing, rubbing, and RNA injection. Results indicated that all three methods cause infection of the pistachio rootstocks with HSVd. However, the slashing inoculation showed higher transmission efficiency in Badami and UCB-1 at the rate of 86.67 and 80 percent, respectively. According to this study, it seems that HSVd infection is increasing on pistachio trees in Kerman province due to lack of attention to sanitary measures during agricultural operations such as pruning, which leads to transmission of disease agents like viroids.