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Beef from Japanese Black cattle (JBK), is popular in Japan and valued for its highly marbled fat content. In JBK, genes affecting oleic acid content in meat have been studied mainly to lower the fat melting point and improve tenderness; however, there has been no direct correlation demonstrated between beef taste and oleic acid. To investigate genes affecting other fatty acids other than oleic acid, polymorphisms of the fatty acid desaturase 2 (FADS2) gene were genotyped and associations with fatty acid profile in JBK beef were investigated. Amplifications of 5’-flanking regions, 12 exons, and 3’-untranslated regions of the FADS2 gene in three Japanese and five Western cattle breeds via PCR, were amplified, sequenced and SNPs were identified using specific TaqMan genotyping assay. Fatty acid composition of intramuscular adipose tissue of the Trapezius muscle was analyzed in JBK steers. Six of the 15 identified SNPs are novel and have never been registered in any public bovine SNP database. A non-synonymous SNP (rs211580559; C > T; 294 Ala > Val) in exon 7 was examined in order to evaluate its association with fatty acid profiles. The data showed that highly significant association existed between rs211580559 and C18:2 (n-6) composition, and accounted for 22.3% of the variation. There were no significant relationships between rs2115-80559 and the other fatty acids. It was concluded that rs211580559 of the FADS2 gene may be a useful selection marker for reducing unfavorable volatiles generated from linoleic acid in JBK beef during the cooking process.