Heat stress is one of abiotic stress that has deleterious effects on crops yield. Therefore, this study aims are to evaluate heat-tolerant maize lines and characterizing them by DNA molecular markers. Inbred lines were generated for two generations by selfing pollination. They were evaluated in each generation for the performance of some agro-morphological traits under normal and heat stress conditions. All the traits of the S₁ and S₂ inbred lines varied significantly among them under both conditions, except plant height was not significant under heat stress for the S₁ lines. The L6 inbred line had the highest yield under the stress conditions in both S₁ andS₂ generations. Moreover, the heat susceptible index showed that the lines, L6 and L40 were the highest tolerant in the both generations. Furthermore, cluster analysis based on morphological traits for the 5 selected S₂ inbred lines could be able to isolate the worst S₂ inbred line under heat stress conditions in an independent cluster. In addition, they were characterized by ISSR and SRAP molecular markers. The ISSR detected higher polymorphism (79.79%) than SRAP marker (58.46%). The ISSR clustering patterns managed to classify the highest yield line (L6) under the heat stress in a separated cluster, but both the SRAP and combined isolated the worst line (L32) in one cluster. The Mantel's test showed a positive correlation among all the studied markers. Additionally, the correlation was significant and highly strong (r=0.915) between morphological traits under normal conditions and SRAP marker. However, the identified S₂ inbred lines with resistance to heat tolerance could be a beneficial source in the development of heat-tolerant maize hybrids.