Conjugated linolenic acid (CLnA) isomers refer to a group of positional and geometric isomers of the omega-3 essential fatty acid – α-linolenic acid (cis-9,cis-12,cis-15 C18:3, ALA). CLnA isomers can be either cis- and/or trans- and their double bonds are separated by a single bond. Food products from ruminants and some plant products (e.g., pomegranate or bitter melon seeds) are the major sources of CLnA isomers for humans. CLnA isomers in ruminants arise as a result of bacterial isomerization of ALA in the rumen. The concentration of CLnA isomers in seed oils is higher than in milk and edible parts of ruminant carcass. The CLnA isomers from the plant sources are in the form of conjugated trienes, whereas those in ruminant products are of conjugated diene type. Some plant seed oils are the richest natural sources of CLnA isomers. So searching for methods of increasing the CLnA isomer content in food of animal origin not exhibiting negative effects on animal welfare and physiology is very important for researchers. A presence of long-chain and very long-chain conjugated unsaturated fatty acids was also confirmed in some ruminant tissues. Clinical studies documented that health-promoting properties have been attributed to CLnA isomers. It was also evidenced that animal diet may influence the CLnA synthesis. The proper identification of geometric and positional isomers of conjugated unsaturated fatty acids in biological samples is a great analytical challenge. Therefore, silver-ion high-performance liquid chromatography with photodiode detection and capillary gas-chromatography (GC) offer the best analysis of these isomers with complementary identification by GC-mass spectrometry.