Global warming poses a threat to rice production. Breeding heat-tolerant rice is an effective and economical approach to addressing this challenge. African rice is a valuable genetic resource for developing heat-tolerant crops due to its intricate mechanism for adapting to high temperatures. Oryza longistaminata, a wild rice species widely distributed in Africa, may harbor an even richer gene pool for heat tolerance, which remains untapped. In this study, we identified three heat-tolerant QTLs from O. longistaminata at the seedling stage. Among these, qTT4 and qTT5 are novel heat-tolerant loci identified in O. longistaminata. Our findings demonstrated that the O. longistaminata alleles for these two QTLs can enhance the heat tolerance of rice seedlings. Remarkably, qTT5 was mapped to a region spanning approximately 287.2 kb, containing 46 expressing genes. Through the analysis of Gene Ontology and expression differences under heat induction, we identified four candidate genes. Our results lay the foundation for the discovery of heat-tolerant genes underlying O. longistaminata and the development of new genetic resources for heat tolerant rice breeding.