Dairy products are the main source of conjugated linoleic acid (CLA), a functional food component with health benefits. The major source of cis-9, trans-11 CLA in milk fat is endogenous synthesis via delta9-desaturase from trans-11 18:1, with the remainder from incomplete rumen biohydrogenation of linoleic acid. Diet has a major influence on milk fat CLA; however, effects of physiological factors have received little attention. Our objectives were to examine milk fat content of CLA and the CLA-desaturase index with regard to: 1) effect of breed, parity, and stage of lactation, and 2) variation among individuals and the relationship to milk and milk fat. Holstein (n = 113) and Brown Swiss (n = 106) cows were fed a single diet and milk sampled on the same day to avoid confounding effects of diet and season. Frequency distributions demonstrated that milk fat content of CLA and CLA-desaturase index varied over threefold among individuals, and this needs to be considered in the design of experiments. Holsteins had a higher milk fat content of CLA and CLA-desaturase index, but breed differences were minor. Parity and days in milk also had little or no relationship to the individual variation for these two CLA variables. Breed, parity, and days in milk accounted for < 0.1, < 0.3, and < 2.0% of total variation in CLA concentration in milk fat, respectively. Milk fat content of CLA and CLA-desaturase index were essentially independent of milk yield, milk fat percent, and milk fat yield. We speculate that the basis for the genetic variation among individuals is related to rumen output of trans-11 18:1 and to a lesser extent cis-9, trans-11 CLA, and to the tissue amount and activity of delta9-desaturase.