Genetic factors are known to influence meat quality in beef cattle. Expression of fatty acid desaturase 2 (FADS2) and fatty acid-binding protein 4 (FABP4) genes in the fat metabolism pathway has been reported to influence fat-related traits. In this study, we aimed to discover DNA polymorphisms in the promoter regions of the FADS2 and FABP4 genes in cattle populations to develop genetic markers. As a consequence, one single nucleotide polymorphism (SNP) in the FADS2 promoter (FADS2 g.-823G>A) and two linked SNPs in the FABP4 promoter (FABP4 g.-295A>G and FABP4 g.-287A>G) were identified. All of them were assumed to influence binding sites for transcription factors, suggesting these SNPs influence the genes׳ expression and beef quality. Therefore, we performed association analysis between the genotypes and traits. Our results revealed that the FADS2 g.-823G>A had significant effects on several traits including beef marbling score (BMS). BMS of the G/G animals in a Holstein population (2.36) was higher than that of the G/A animals (2.13). The same tendency was observed in Japanese Black populations. On the other hand, although the FABP4 SNPs also had significant effects on some traits, they did not show additive effects on any of the traits analyzed in this study. Our results suggest that the FADS2 g.-823G>A is a useful genetic marker for breeding in beef cattle.