Stearoyl-CoA desaturase-1 (SCD1) is a key enzyme in the biosynthesis of monounsaturated fatty acids and is considered a candidate gene for improving milk and meat quality traits. Sanger sequencing was employed to investigate the genetic polymorphism of the fifth exon and intron of bovine SCD1, revealing four SNPs, g.21272246 A>G, g.21272306 T>C, g.21272422 C>T, and g.21272529 A>G. Further variance analysis and multiple comparisons were conducted to examine the relationship between variation sites and economic traits in Chinese Simmental cattle, as well as milk production traits in Holstein cows. The findings revealed these four loci exhibited significant associations with carcass traits (carcass weight, carcass length, backfat thickness, and waist meat thickness), meat quality (pH value, rib eye area, and marbling score), adipogenic traits (fat score and carcass fat coverage rate), and fatty acid composition (linoleic acid and α-linolenic acid). Furthermore, these loci were additionally found to be significantly associated with average milk yield and milk fat content in cows. In addition, a haplotype analysis of combinations of SNPs showed that H2H3 has a significant association with adipogenic traits and H2H2 was associated with higher levels of linoleic acid and α-linolenic acid than the other combinations. These results suggest that the four SNPs are expected to be prospective genetic markers for the above economic traits. In addition, the function of SNPs in exon 5 of SCD1 on gene expression and protein structure needs to be explored in the future.