This study aimed to evaluate the fatty acid composition, including the contents, of conjugated linoleic acid cis9trans11 C18:2 (CLA) and trans C18:1 and C18:2 isomers in hard cow, sheep, and goat cheeses found on the Polish market and to compare lipid quality indices in these cheeses. The gas chromatography method was used to determine the fatty acid profile. The study demonstrated various contents of cis9trans 11 C18:2 (CLA), trans C18:1, and C18:2 isomers and the values of lipid quality indices in the cheeses. Sheep and goat cheeses were richer sources of short-chain fatty acids (SCFA) (14.73 ± 2.55% and 14.80 ± 2.80%, respectively) than the cow cheeses (9.38 ± 0.87%). The cow cheeses had a significantly higher (p < 0.05) content of monounsaturated fatty acids (MUFA), the lowest n-6/n-3 ratio, and the highest content of fatty acids, inducing a desirable dietary effect in humans (DFA) compared to the sheep and goat cheeses. Significantly higher (p < 0.05) contents of polyunsaturated fatty acids (PUFA) were found in sheep cheeses. Goat cheeses had the highest n-3 PUFA content and the lowest values of the thrombogenicity index (TI) (2.67 ± 0.44) compared to the sheep and cow cheeses (3.14 ± 0.29 and 3.13 ± 0.13, respectively). The cow, sheep, and goat cheeses were characterized by similar values of the hypocholesterolemic/hypercholesterolemic (H/H) ratio. Sheep cheeses had the highest levels of cis9trans11 C18:2 (CLA) and the highest total content of trans C18:1 and trans C18:2 isomers. The research showed that sheep, cow, and goat cheeses offered various health benefits. The differences in fatty acid composition and the different values of the lipid quality indices found in the cheeses may be due to differences in both the composition of milk used to produce them and the cheese-making technology. Studies conducted by many authors have indicated that the feeding system of the ruminants has a significant impact on the quality and chemical composition of milk, as well as its applicability for cheese production.

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