In this paper, the seedlings of 27 inbred lines of bitter gourd with four leaves and one heart were used as materials. After pre-cultivated one day, the seedlings were subjected to 39 ℃ temperature stress. And the 27 germplasm resources were divided into three types: high heat resistance, medium heat resistance and heat sensitivity screened by the heat damage index (TDG) after 72 hours of high temperature stress. Among them, six inbred lines with different heat tolerance were selected to measure 12 morphological and physiological indexes subjected to 39 ℃ stress for 0 h and 48 h, respectively, including plant height (PH), stem diameter (SD), plant fresh matter (SFW) and dry matter (SDW) and leaf relative chlorophyll content (SPAD), electrical conductivity (REL), peroxidase activity (POD), catalase activity (CAT), superoxide dismutase activity (SOD), malondialdehyde content (MDA)), proline content (Pro) and soluble sugar content (SS). The comprehensive evaluation values were obtained by the principal component analysis of heat resistance coefficients (α) of 12 morphological and physiological indexes to evaluate heat resistance of the seedlings. The mathematical model of heat resistanc evaluation was established by the stepwise regression analysis to obtain the heat resistance evaluation index system of bitter gourd. According to TDG, among the 27 bitter gourd inbred lines, three were heat-resistant, 18 were medium-heat-resistant and six were heat-sensitive. A mathematical evaluation model of heat tolerance of bitter gourd was established using the stepwise regression equation, which is Y=–0.426+0.049X₁–0.011X₂–0.009X₃–0.018X₄. The established optimal regression equation was used to predict the heat resistance of the tested materials, and the predictive values were basically consistent with the comprehensive heat tolerance value. Therefore, the heat resistance coefficients of SFW (X₁), POD activity (X₂), CAT activity (X₃) and SS content (X₄) could rapidly and accurately estimate bitter gourd heat resistance and were suggested to be used as evaluation indexes for heat resistance of bitter gourd. In order to provide a basis for the breeding of new heat-resistant bitter gourd varieties in Hainan in summer season, the heat-resistant bitter gourd germplams were screened and a heat-resistant evaluation system was constructed.