The objective of this study was to estimate genetic parameters for conjugated linoleic acid (CLA) and other selected milk fatty acid (FA) content and for unsaturation ratios in the Italian Holstein Friesian population. Furthermore, the relationship of milk FA with milk fat and protein content was considered. One morning milk sample was collected from 990 Italian Holstein Friesian cows randomly sampled from 54 half-sib families, located in 34 commercial herds in the North-eastern part of Italy. Each sample was analyzed for milk percentages of fat and protein, and for single FA percentages (computed as FA weight as a proportion of total fat weight). Heritabilities were moderate for unsaturated FA, ranging from 0.14 for C16:1 to 0.19 for C14:1. Less than 10% of heritability was estimated for each saturated FA content. Heritability for index of desaturation, monounsaturated FA and CLA/trans-11 18:1 ratio were 0.15, 0.14, and 0.15, respectively. Standard errors of the heritability values ranged from 0.02 to 0.06. Genetic correlations were high and negative between C16:0 and C18:0, as well as between C14:0 and C18:0. Genetic correlations of index of desaturation were high and negative with C14:0 and C16:0 (-0.70 and -0.72, respectively), and close to zero (0.03) with C18:0. The genetic correlation of C16:0 with fat percentage was positive (0.74), implying that selection for fat percentage should result in a correlated increase of C16:0, whereas trans-11 C18:1 and cis-9, trans-11 C18:2 contents decreased with increasing fat percentage (-0.69 and -0.55, respectively). Genetic correlations of fat percentage with 14:1/14 and 16:1/16 ratios were positive, whereas genetic correlations of fat percentage with 18:1/18 and CLA/trans-11 18:1 ratios were negative. These results suggest that it is possible to change the milk FA composition by genetic selection, which offers opportunities to meet consumer demands regarding health aspects of milk and dairy products.