Croftonweed originated from Mexico is a worldwide notorious invasive weed. The objectives of this study were to screen heat tolerance in different croftonweed populations, determine the effect of heat stress on two photosystems and probe the mechanism of acquired heat tolerance. According to conventional tests of plant injury and fast chlorophyll fluorescence rise kinetics, four different croftonweed populations collected from South China were successfully classified into three categories by exposing whole plants to heat treatment at 40°C: sensitive, intermediate, tolerant. Evidence from the JIP-test indicated that inhibition of the oxygen evolution complexes (OEC) and inactivation of PSII reaction centers (RCs) were the primary cause of heat damage. In mild heat stress (<40°C), slightly damaged the OEC without creating a visible K-step in the fluorescence rise OJIP curve. In moderate heat stress or stronger (≥40°C), a pronounced K-step due to irreversible severe damage on the OEC occurred. Additionally, inactivation of PSII RCs, down-regulation of energetic connectivity of PSII units, destruction of PSII antenna architecture, losing overall photosynthetic activity of PSII, increase of PSI activity also took place. Furthermore, the tolerant population had lesser damage degree on photosynthetic capacity relative to intermediate and sensitive populations. Finally, a reliable model, based on the most sensitive parameter PIABS and VK as a characteristic parameter for heat stress, is presented for ranking and identifying heat tolerance in different croftonweed populations. The heat sensitivity index (HSI) is also introduced as an indicator of plant heat sensitivity. The smaller the HSI value is, the higher the level of tolerance to heat stress is. We also found that the tolerance degree of four croftonweed populations to heat stress is significantly correlated to the extreme high temperature. This indicates that acquired heat tolerance in croftonweed populations results from plant adaptation to ambient high temperatures. Acquirement of heat tolerance confers a possible risk for croftonweed to spread further to currently hotter areas.