Muscle characteristics such as myofiber diameter, density, and total number are important traits in broiler breeding and production. In the present study, 19 SNP of 13 major genes, which are located in the vicinity of quantitative trait loci affecting breast muscle weight, including INS, IGF2, PIK3C2A, AKT3, PRKAB2, PRKAG3, VEGFA, RPS6KA2/3, FIGF, and TGF-β1/2/3, were chosen to be genotyped by high-throughput matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in a broiler population. One hundred twenty birds were slaughtered at 6 wk of age. Body weight, breast muscle weight, myofiber diameter, density, and total number were determined for each bird. Six SNP with a very low minor allele frequency (<1%) were excluded for further analysis. The remaining 13 SNP were used for the association study with muscle characteristics. The results showed that SNP in TGF-β1/2/3 had significant effects on myofiber diameter. A SNP in PRKAG3 had a significant effect on myofiber density (P < 0.05). A C > G mutation in FIGF was strongly associated with total fiber number (P < 0.05). Additionally, birds with the GG genotype of the C > G mutation in AKT3 had significantly larger myofiber numbers (P < 0.05) than birds with the CC or GC genotype. The SNP identified in the present study might be used as potential markers in broiler breeding.