Background Wet direct-seeded rice is a possible alternative to conventional puddled transplanted rice; the former uses less water and reduces labor requirements. Improving seed reserve utilization efficiency (SRUE) is a key factor in facilitating the application of this technology. However, the QTLs controlling this trait are poorly investigated. In this study, a genome-wide association study (GWAS) was conducted using a natural population composed of 542 accessions of rice (Oryza sativa L.) which were genotyped using 266 SSR markers. Large phenotypic variations in SRUE were found in the studied population. Results The average SRUE over 542 accessions across two years (2016 and 2017) was 0.52 mg.mg− 1, ranging from 0.22 mg.mg-1 to 0.93 mg.mg− 1, with a coefficient of variation of 22.66%. Overall, 2879 marker alleles were detected in the population by 266 pairs of SSR markers, indicating a large genetic variation existing in the population. Using general linear model method, 13 SSR marker loci associated with SRUE were detected and two (RM7309 and RM434) of the 13 loci, were also detected using mixed linear model analyses, with percentage of phenotypic variation explained (PVE) greater than 5% across two years. The 13 association loci (P < 0.01) were located on all chromosomes except chromosome 11, with PVE ranging from 5.05% (RM5158 on chromosome 5) to 12% (RM297 on chromosome 1). Association loci RM7309 on chromosome 6 and RM434 on chromosome 9 revealed by both models were detected in both years. Twenty-three favorable alleles were identified with phenotypic effect values (PEV) ranging from 0.10 mg.mg− 1 (RM7309–135 bp on chromosome 9) to 0.45 mg.mg− 1 (RM297–180 bp on chromosome 2). RM297–180 bp showed the largest phenotypic effect value (0.44 mg.mg− 1 in 2016 and 0.45 mg.mg− 1 in 2017) with 6.72% of the accessions carrying this allele and the typical carrier accession was Manyedao, followed by RM297–175 bp (0.43 mg.mg− 1 in 2016 and 0.44 mg.mg− 1 in 2017). Conclusion Nine novel association loci for SRUE were identified, compared with previous studies. The optimal parental combinations for pyramiding more favorable alleles for SRUE were selected and could be used for breeding rice accessions suitable for wet direct seeding in the future.