Rumex thyrsiflorus Fingerh. is a dioecious species with heteromorphic sex chromosomes. Due to sexual dimorphism and the content of bioactive constituents used in pharmacology, this species is an interesting object of study. A complex analysis of selected physiological and biochemical aspects of the sex-related response to heat stress in vitro and in vivo was carried out. The experiment included in vitro regenerated plants and plants obtained from seeds. Regenerants were obtained from hypocotyls on Murashige and Skoog (MS) medium supplemented with 2.27 μM thidiazuron (TDZ). The sex of the plants was determined by molecular analysis based on genetic sex markers. Analysis of the main photosynthetic parameters indicated that in vitro regenerated plants showed a decrease in photosystem II (PSII) activity when directly exposed to a stressor. However, in contrast to the seed-derived plants, they adapted efficiently to the recovery conditions within 1 week after the stress was terminated. Furthermore, in vitro regenerated male and female plants acclimatised well to field conditions and showed greater stress tolerance based on better efficiency of the photosynthetic apparatus and the highest chlorophyll a/b ratio. In case of plants derived from seeds, male plants were less sensitive to heat stress and showed greater stability of PSII at high temperatures compared to female plants. The results showed that the response to high-temperature stress depends on the sex and the origin of the plants, i. e. in vitro regenerated plants versus plants obtained from seeds. Even short-term heat stress resulted in differences in photosynthetic efficiency. Biochemical analysis of antioxidant activity in response to heat stress, carried out for the first time in Rumex thyrsiflorus, has allowed the identification of the following forms of superoxide dismutase (SOD): manganese (MnSOD), iron (FeSOD) and two copper-zinc isoforms (Cu/ZnSOD I and Cu/ZnSOD II).