The objective of this study was to evaluate the utilization of different sources of unsaturated long-chain fatty acids in diets for dairy cows during the transition period and early lactation on the milk fatty acid profileandcompositionoftheprotein fraction. Thirty-six Holstein cows were divided into three groups, fed the following diets: control (C); soybean oil (SO); and calcium salts of long-chain unsaturated fatty acids (CS). The milk samples utilized for analysis were obtained weekly from parturition to twelve weeks of lactation; each one of the samples originated from two daily milkings. Milk composition and total nitrogen, non-protein nitrogen and non-casein nitrogen levels were analyzed. The cows receiving the diet with calcium salts had lower concentrations of non-protein nitrogen (%CP) in milk compared with the animals fed the diet with soybean oil. There was a decrease in concentration of medium-chain fatty acids C12-C16, and a concomitant increase in concentrations of long-chain fatty acids >C18 in milk fat for the animals fed the diets CS and SO when compared with diet C. Soybean oil and CS diets increased milk-fat concentrations of the acids C18: 1 trans-11, C18: 2 cis-9, trans-11 and C18: 2 trans-10 cis-12 in relation to diet C. The utilization of sources of long-chain fatty acids in the diet of dairy cows increases the biological value of milk in early lactation due to higher concentrations of specificfattyacidssuchasCLAC18:2cis-9,trans-11.