Bilal, G., Cue, R. I., Mustafa, A. F. and Hayes, J. F. 2014. Effects of parity, age at calving and stage of lactation on fatty acid composition of milk in Canadian Holsteins. Can. J. Anim. Sci. 94: 401-410. The objectives of the present study were to study the effects of parity, age at calving and stage of lactation on individual fatty acids and fatty acid unsaturation indices in milk of Canadian Holsteins. One morning milk sample was collected from each of 3185 dairy cows between February and June 2010 from 52 commercial herds enrolled in the Quebec Dairy Production Centre of Expertise, Valacta. Individual fatty acid proportions (g 100 g^-1 of total fatty acids) were determined for each sample by gas chromatography. Editing for registration status, cow, sire and dam identification, breed, country, age at calving and stage of lactation reduced the data set to 2573 cows representing 46 herds. The statistical model included the fixed effects of parity, age at calving and stage of lactation each nested within parity and random effects of herd-year-season of calving and residual. First-parity cows had relatively higher proportions of beneficial unsaturated fatty acids, from a human health standpoint, and lower proportions of potentially undesirable saturated fatty acids in milk fat as compared with later parity cows. Milk fatty acid profile varied with stage of lactation. The proportions of short- and medium-chain fatty acids (C6:0 to C14:0) were low in the beginning of lactation and increased during the early part of lactation. Long-chain fatty acids (especially C18:0 and oleic acid) and unsaturation indices (C16, C18 and total) showed an opposite trend to that of short- and medium-chain fatty acids. The study showed that parity and lactation stage contribute to variation in bovine milk fat composition.