Frequency-dependent selection (FDS) is an evolutionary regime that can maintain or reduce polymorphisms. Despite the increasing availability of polymorphism data, few effective methods are available for estimating the gradient of FDS from the observed fitness components. We modeled the effects of genotype similarity on individual fitness to develop a selection gradient analysis of FDS. This modeling enabled us to estimate FDS by regressing fitness components on the genotype similarity among individuals. We detected known negative FDS on the visible polymorphism in a wild Arabidopsis and damselfly by applying this analysis to single-locus data. Further, we simulated genome-wide polymorphisms and fitness components to modify the single-locus analysis as a genome-wide association study (GWAS). The simulation showed that negative or positive FDS could be distinguished through the estimated effects of genotype similarity on simulated fitness. Moreover, we conducted the GWAS of the reproductive branch number in Arabidopsis thaliana and found that negative FDS was enriched among the top-associated polymorphisms of FDS. These results showed the potential applicability of the proposed method for FDS on both visible polymorphism and genome-wide polymorphisms. Overall, our study provides an effective method for selection gradient analysis to understand the maintenance or loss of polymorphism.