Stigma characteristics are important factors affecting the seed yield of hybrid rice per unit area. Natural variation of stigma characteristics has been reported in rice, but the genetic basis for this variation is largely unknown. We performed a genome-wide association study on three stigma characteristics in six environments using 1.3 million single-nucleotide polymorphism (SNPs) characterized in 353 diverse accessions of Oryza sativa. An abundance of phenotypic variation was present in the three stigma characteristics of these collections. We identified four significant SNPs associated with stigma length, 20 SNPs with style length (SYL), and 17 SNPs with the sum of stigma and style length, which were detected repeatedly in more than four environments. Of these SNPs, 28 were novel. We identified two causal gene loci for SYL, OsSYL3 and OsSYL2; OsSYL3 was co-localized with the grain size gene GS3. The SYL of accessions carrying allele OsSYL3AA was significantly longer than that of those carrying allele OsSYL3CC. We also demonstrated that the outcrossing rate of female parents carrying allele OsSYL2AA increased by 5.71% compared with that of the isogenic line carrying allele OsSYL2CC in an F1 hybrid seed production field. The allele frequencies of OsSYL3AA and OsSYL2AA decreased gradually with an increase in latitude in the Northern Hemisphere. Our results should facilitate the improvement in stigma characteristics of parents of hybrid rice.