In this research two-dimensional GC was used to analyse, for the first time, the detailed fatty acid (FA) profiles of 11 dairy matrices: raw milk (evening whole, evening partially skimmed, morning whole, and vat milk), cream, fresh cheese, whey, ricotta, scotta, 6- and 12-month-ripened cheeses, obtained across artisanal cheese- and ricotta-making trials carried out during the summer period while cows were on highland pastures. Samples were collected during 7 cheese- and ricotta-making procedures carried out at 2-week intervals from bulk milk to study possible differences in the transfer and modification of FA. Compared with morning milk, evening milk had fewer de novo synthetised FA. The detailed FA profile of partially skimmed milk differed little from that of evening whole milk before skimming, but the cream obtained differed from partially skimmed milk and from fresh cheese in about half the FA, due mainly to higher contents of all de novo FA, and lower contents of n-3 and n-6 FA. Fresh cheese and whey had similar FA profiles. The ricotta manufacturing process affected the partition of FA between ricotta and scotta, the FA profile of the latter differing in terms of groups and individual FA from the former, whereas ricotta and fresh cheese had similar composition of FA. In general, there was an increase in medium-chain saturated FA, and a decrease in many polyunsaturated FA during the first 6 months of ripening, but not during the second 6 months. Two-dimensional GC yielded a very detailed and informative FA profile on all the 11 dairy products and by-products analysed.