AbstractRice consumption is limited by being a carbohydrate‐dense food with a high glycemic index (GI). Rice GI can be reduced by increasing its resistant starch (RS) content, which resists digestion in the gastrointestinal tract. To identify novel genes and gene combinations useful for developing high‐RS rice varieties, we identified a novel starch mutant (GSOR 610001), mapped it to the soluble starch synthase IIIa (ssIIIa) locus, and evaluated its impact on RS individually and in combination with two additional genes known to affect RS production: Wx and BEIIb. Of the three genes, the beIIb allele increased RS the most, followed by Wxa. The new ssIIIa mutant increased RS to a lesser degree when genetically combined with the high‐amylose Wxa allele and the non‐mutant BEIIb allele; instead, ssIIIa+beIIb double mutants decreased RS indicating the two mutant genes act antagonistically. The higher RS of beIIb was associated with its amylopectin having higher proportion of long and degree of polymerization (DP) 19–37 chains and less branching than the wild type. The ssIIIa mutant had lower long amylopectin chains than the wild type but maintained its amylopectin branched structure, which prevented short‐term RS formation after cooking. Increased RS from ssIIIa was associated with amylose content. The antagonistic effect of adding ssIIIa to beIIb was due to the reduction of less‐branched, long amylopectin chains of beIIb. In summary, the target genotype that optimized RS was Wxa+beIIb+SSIIIa (averaged 6.5 ± 0.8% RS [milled basis]), and the validated molecular markers will speed development of rice varieties having high RS/low GI using marker‐assisted selection.