The abundance of 20- to 24-carbon fatty acids in omasal digesta of cows fed grass silage-based diets supplemented with 0 (Control) and 250 g/day of fish oil (FO) was examined to investigate the fate of long-chain unsaturated fatty acids in the rumen. Complimentary argentation thin-layer chromatography and gas-chromatography mass-spectrometry analysis of fatty acid methyl esters and corresponding 4,4-dimethyloxazoline derivatives prepared from fish oil and omasal digesta enabled the structure of novel 20- to 22-carbon fatty acids to be elucidated. Compared with the Control, the FO treatment resulted in the formation and accumulation of 27 novel 20- and 22-carbon biohydrogenation intermediates containing at least one trans double bond and the appearance of cis-14 20:1, 20:2n-3, 21:4n-3 and 22:3n-6 not contained in fish oil. No conjugated ≥ 20-carbon fatty acids were detected in Control or FO digesta. In conclusion, fish oil in the diet results in the formation of numerous long-chain biohydrogenation intermediates in the rumen of lactating cows. Comparison of the intake and flow of 20-, 21- and 22-carbon fatty acids at the omasum in cows fed the Control and FO treatments suggests that the first committed steps of 20:5n-3, 21:5n-3 and 22:6n-3 hydrogenation in the rumen involve the reduction and/or isomerisation of double bonds closest to the carboxyl group.