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Abstract Low-temperature germinability (LTG) is an important agronomic trait that can affect the planting time, planting area, and grain yield of staple crops, such as rice. However, the genetic mechanism of LTG is still unclear. In this study, a multi-parental permanent population with 208 single segment substitution lines (SSSLs) was used to conduct a genetic dissection for LTG across four cropping seasons. LTG was a typical quantitative trait with a high combined broad-sense heritability of 0.71. By comparison with the recipient parent, Huajingxian74, 24 SSSLs were identified as carrying LTG QTLs, which were further merged into integrated QTLs with shorter genetic distances by substitution mapping. Finally, 14 LTG QTLs were mapped on ten chromosomes, including seven positive-effect and seven negative-effect QTLs, with additive effect contributions ranging from 19.2 to 39.9%. qLTG3a, a main-effect and novel QTL, was confirmed by bulk segregant analysis using an F2 segregating population, and five key recombinants were selected to develop F3 populations for progeny testing. Marker-trait association analysis fine mapped qLTG3a to a 332.7-kb physical region between markers M6026 and M6341. Within this interval, 40 annotated genes were revealed, and three genes (Os03g0213300, Os03g0214400, and Os03g0214600) were considered as pivotal candidate genes for qLTG3a based on their sequence variations and expression patterns. Besides low temperature, qLTG3a can also enhance seed germination under standard temperature and osmotic stress. In summary, this study identified some genetic factors regulating LTG and opened a new window for breeding elite direct-seeded rice varieties. It will help reduce the climate risk in the production process of rice, which is of great significance to ensuring food security.