Chibisa, G. E., Christensen, D. A. and Mutsvangwa, T. 2013. Replacing canola meal as the major protein source with wheat dried distillers' grains alters omasal fatty acid flow and milk fatty acid composition in dairy cows. Can. J. Anim. Sci. 93: 137-147. Wheat dried distillers' grains with solubles (W-DDGS) has a high fat content and is now commonly fed to dairy cows in western Canada. The objective was to determine the effects of replacing canola meal (CM) with W-DDGS on omasal fatty acid (FA) flow and milk FA composition. Four ruminally-cannulated lactating dairy cows were used in a 4×4 Latin square design with 28-d periods. Cows were fed either a standard barley silage-based total mixed ration (TMR) (0% W-DDGS, control) or TMR containing 10, 15 and 20% W-DDGS, with W-DDGS replacing CM as the major protein source. The omasal flow of C18:1 trans-10, C18:1 trans-11, C18:2n-6, C18:3n-3, conjugated linoleic acid (CLA) cis-9, trans-11, and total CLA increased linearly with increasing levels of W-DDGS. There were quadratic changes in milk concentrations of total C18:2, C18:3n-3, total n-3 polyunsaturated fatty acid (PUFA), and total n-6 PUFA as the level of W-DDGS increased. Feeding increasing amounts of W-DDGS resulted in a linear increase in milk concentrations of CLA cis-9, trans-11, CLA trans-11, trans-13, and total CLA, whereas milk concentrations of C18:1 trans-11 tended to increase linearly when level of W-DDGS increased. Feeding increasing amounts of W-DDGS resulted in quadratic changes in milk concentrations of C18:2n-6 and C18:3n-3. In summary, feeding increasing amounts of W-DDGS increased milk concentrations of key FA like C18:2n-6, C18:3n-3, CLA cis-9, trans-11, and total CLA, which can potentially improve human health.