The myostatin gene (MSTN), which encodes the protein myostatin, is pleiotropic, and its expression has been associated with both increased and decreased adipogenesis and increased skeletal muscle mass in animals. In this study, the polymerase chain reaction, coupled with single strand conformation polymorphism analysis, was utilized to reveal nucleotide sequence variation in bovine MSTN in 410 New Zealand (NZ) Holstein-Friesian × Jersey (HF × J)-cross cows. These cows ranged from 3 to 9 years of age and over the time studied, produced an average 22.53 ± 2.18 L of milk per day, with an average milk fat content of 4.94 ± 0.17% and average milk protein content of 4.03 ± 0.10%. Analysis of a 406-bp amplicon from the intron 1 region, revealed five nucleotide sequence variants (A-E) that contained seven nucleotide substitutions. Using general linear mixed-effect model analyses the AD genotype was associated with reduced C10:0, C12:0, and C12:1 levels when compared to levels in cows with the AA genotype. These associations in NZ HF × J cross cows are novel, and they suggest that this variation in bovine MSTN could be explored for increasing the amount of milk unsaturated fatty acid and decreasing the amount of saturated fatty acid.