To investigate the genetic factors that affect fatty acid composition of beef, we compared the full-length bovine stearoyl-CoA desaturase (SCD) cDNA from 20 Japanese Black steers. Two types of the SCD gene with single nucleotide polymorphisms (SNPs) were observed in the ORF of SCD cDNA, in which an amino acid replacement from valine (type V) to alanine (type A) was predicted. We developed a method for genotyping these two SCD genes based on PCR-RFLP. We have classified 1003 Japanese Black carcasses into three genotypes, VV, VA, and AA, and compared fatty acid composition among them. The SCD type A gene contributed to higher MUFA percentage and lower melting point in intramuscular fat. The SCD genotype was not the only genetic factor contributing to fatty acid composition of Japanese Black steers, but the SCD genotype was considered one of the causes of genetic variation in fatty acid composition of Japanese Black steers. Transcription factors such as sterol regulatory element binding protein-1c (SREBP-1c) may account for the remaining part of the genetic variation in fatty acid composition.