Tartary buckwheat (Fagopyrum tataricum Gaertn, TB) is an annual multigrain crop belonging to the genus Fagopyrum within the Polygonaceae family. TB is valued for its balanced nutrient composition and high flavonoid content, particularly rutin, which possesses various health and medicinal benefits. Therefore, rutin has become a critical trait in determining the quality of TB. The rutin content (RC) of 101 natural TB materials measured in this study ranged from 14.08 to 24.50 mg/g, with an average value of 19.46 mg/g. This result revealed substantial natural variation in RC within the TB population. Furthermore, 314,166 high-quality single nucleotide polymorphisms (SNPs) were identified through whole-genome resequencing. Population structure analysis demonstrated that genetic information was consistent among the samples, while significant structural differences were observed among the groups. A genome-wide association study (GWAS) employing a mixed linear model (MLM) identified the SNP site Ft4:1777524 as significantly associated with rutin levels. Within the 400 kb candidate region, 27 key genes were identified. Further joint analysis of previous transcriptome data revealed that FtS1Fa1 is closely linked to RC. Molecular biological characterization revealed that this gene belongs to the S1Fa-like family, is localized in chloroplasts, but lacks transcriptional activation capabilities. Functional studies indicated that FtS1Fa1 can promote rutin accumulation in TB by upregulating the expression of the rutin biosynthesis gene. In conclusion, this study enhances the understanding of the regulatory mechanisms governing rutin biosynthesis in TB and provides important germplasm resources for the molecular breeding of high-rutin TB varieties.