Abstract Alkali stress is one of the most serious abiotic stresses limiting crop yield and it has become an increasingly serious global problem in recent years. Alkalinity tolerance (AT) at the seedling stage is one of the determinant factors for establishment of rice population under alkaline stress condition. Here, we evaluated and measured seven traits related to AT of 528 diverse rice accessions at the seedling stage. Xian accessions were generally more alkali-tolerant than Geng accessions. GJ-tmp accessions showed the most alkali tolerance in the Geng subgroups and XI-1B accessions had the weakest alkali tolerance in the Xian subgroups. A total of 121 QTLs were identified for AT by genome-wide association study (GWAS), and five important candidate genes, LOC_Os01g19800, LOC_Os01g20160, LOC_Os01g52500, LOC_Os01g67370 and LOC_Os03g03900, were selected by gene function annotation, haplotype analysis, and qRT-PCR. Pyramiding of multiple AT advanced candidate genes is a favorable strategy for improving AT of rice varieties. Our study has screened alkali-tolerant germplasm resources and provided valuable genetic information for alkali-tolerant rice breeding.