Salinity–alkalinity stress is one of the major abiotic stresses that limit rice production in the world. The salinity–alkalinity tolerance of rice at the germination stage has a direct effect on the survival and final yield of seedlings in direct sowing. However, there are few reports of quantitative trait locus (QTL) mapping and mapping-based cloning of alkaline tolerance at the bud burst stage. Here, new alkaline tolerance loci were constructed for F_2:3 and BC₃F₄ by using IR36 and Long-Dao124 (LD124) rice varieties with significant differences in alkaline tolerance. Through linkage analysis and a fine-mapping strategy, qAT3 was identified as the major QTL for alkaline tolerance at the bud burst stage, which could explain 14.79% of the phenotypic variation on average. Then the interval was fine-mapped to 110.265 kb, and the candidate gene LOC_Os03g03150 was predicted by quantitative real-time polymerase chain reaction (qRT-PCR) analysis and sequencing analysis. This provides a key theory for the molecular breeding of alkali-tolerant genes and the study of the molecular mechanism of alkali tolerance in LD124.