In order to clarify the heat tolerance of F5 and F6 inbred lines derived from inter-specific hybrids of Cucurbita moschata (MO) × Cucurbita maximam (MA), the relative electrical conductivity (E) was measured before and after heat stress treatment, followed by the calculation of the rate of injury (RI), positive hyperparental advantage (PHA%) and negative hyperparental advantage (NHA%). The results showed that RI represented higher detection sensitivity and negative correlation with heat tolerance. The outcomes in tested inbred lines were coincident with the phenotyping results. The heat stress treatment resulted in elevated Evalue in inbred lines, suggesting a damage on the leaf cell membrane. The RI value of MO and MA were 0.24±0.10 and 0.65±0.29, respectively. Sixteen and 14 inbred lines showing heat tolerant and sensitive respectively, as well as six with moderate heat tolerance were identified. The heat tolerance trait was polarization among 36 tested inbred lines, and the tolerance enhance effective was obvious. The heat tolerance of three F4 lines were differing. The F5 offspring of 18SH and 18SE showed enhanced heat tolerance, whereas the F5 offspring of 18SF represented decreased heat tolerance. By taking the RI value of 0.15 as the threshold value, 13 heat tolerance inbred lines with higher PHA% value were selected, including ten F5 inbred lines derived from 18SH, two F5 derived from 18SE, and one F6 derived from 18FB. Collectively, these heat-tolerant lines might provide the germplasm basis in breeding for Cucurbita maximam cultivars breeding suitable for over-summer farming.