Identifying Quantitative Trait Loci (QTLs) and candidate genes related to resistant starch (RS) content in rice grains aids in elucidating the genetic mechanisms underlying RS accumulation and facilitates the breeding of rice varieties with high RS content. In this study, 139 rice germplasm resources were utilized as research materials, and their grain RS content was measured over three consecutive years from 2021 to 2023. Combining 255,501 high-quality Single Nucleotide Polymorphisms (SNPs) markers, a genome-wide association study (GWAS) was conducted using the FarmCPU model, revealing a total of 12 QTLs significantly associated with rice grain RS content. Among them, qRS-8-8 was consistently detected in four environments, explaining 33.99% of phenotypic variation, while qRS-11-1 was repeatedly detected in two environments, accounting for 14.06% of phenotypic variation. By annotating gene functions, analyzing differential gene expression levels, and conducting haplotype analysis, two candidate genes related to grain RS content were screened within the repeatedly detected QTL intervals. Specifically, LOC_Os08g28670 encodes a pathogenesis-related/Bet v1-like protein family member, and LOC_Os11g44950 encodes a glycoside hydrolase family 3 protein. The findings of this study provide a theoretical reference for further elucidating the genetic mechanisms of RS content formation in rice grains and contribute to the genetic improvement of rice quality.