The aim of this study was to evaluate the morpho-biochemical reactions of pistachio rootstocks and their interspecific hybrids to frost and freezing temperature stress. The experiment was performed as a factorial in a completely randomized design with three replications. The first factor was temperature (4, 0 and -4 °C) and the second factor was rootstock ((pistachio domestic rootstocks (Badami Zarand, Sarakhs and Qazvini), hybrids (Qazvini×Khinjuk, Qazvini× Mutica, Qazvini × Baneh Baghi, Qazvini × Atlantica, Qazvini× Integerrima), non-domesticated species including Atlantica ((Pistacia atlantica, Integerrima, Khinjuk, Sarakhs, Mutica (P. atlantica sub. mutica) and Baneh Baghi (P. mutica × P. vera) and UCB1 hybrid. The results of this study showed that morphological and biochemical indices were statistically significant by the applied treatments. According to the results, with decreasing the temperature to -4 °C, the percentage and severity of damage significantly increased (p <0.01). The evaluation of the damage severity showed that the Mutica, hybrid Qazvini × Mutica, Sarakhs, Qazvini were tolerant to freezing temperature and the highest damage index was observed in UCB1, Integerrima and Khinjuk. Based on the results, the highest percentage of seedling drought damage was observed in UCB1 rootstock and the highest percentage of shoot tip drought was related to Integerrima and Khinjuk rootstock. The highest activity of Ascorbate peroxidase enzyme was in Sarakhs, Qazvini and Mutica rootstocks and the highest activity of Superoxide dismutase enzyme was related to Mutica rootstock and hybrid Qazvini× Mutica. The interaction effect showed that the activity of Catalase, Ascorbate peroxidase, Superoxide dismutase, and Guaiacol peroxidase enzymes in cold-tolerant rootstocks was significantly higher than cold-susceptible rootstocks. The results of cluster diagrams based on the evaluated indices showed that in cold temperature stresses, Mutica, Qazvini × Baneh, Sarakhs, Qazvini, and Qazvini ×Atlantica were in a subgroup which indicated the close proximity can be considered as the tolerant rootstocks to frost and freezing temperature stress in the breeding program.