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Single nucleotide polymorphisms in the coding region of the bovine stearoyl-CoA desaturase 1 gene have been predicted to result in p.293A (alanine at amino acid 293) and p.293V (valine at amino acid 293) alleles at the stearoyl-CoA desaturase1 locus. The objectives of this study were to evaluate the extent to which genotypes at the stearoyl-CoA desaturase 1 locus and stage of lactation influence milk fatty acid composition in Canadian Holstein cows. Cows with the p.293AA genotype had higher C10 index, C12 index and C14 index and higher concentrations of C10:1 (10 carbon fatty acid with one double bond), C12:1 (12 carbon fatty acid with one double bond) and myristoleic acid (C14:1) compared with the p.293AV or p.293VV cows. Cows had higher C18 index and total index, and lower C10 index, C12 index, C14 index and CLA index during early lactation compared with the subsequent lactation stages. Early lactation was also characterized by higher concentrations of oleic acid (C18:1 cis-9), vaccenic acid (C18:1 trans-11), linoleic acid (C18:2), monounsaturated fatty acids and total polyunsaturated fatty acids, and lower concentrations of capric acid (C10:0), C10:1, lauric acid (C12:0), C12:1, myristic acid (C14:0), myristoleic acid (C14:1), palmitic acid (C16:0) and total saturated fatty acids compared with the subsequent lactation stages. Neither the stearoyl-CoA desaturase 1 genotype nor the stage of lactation had an influence on conjugated linoleic acid concentrations in milk.