Rumen biohydrogenation (BH) of C18:3n-3 (ALA) and C18:2n-6 (LA) has been shown to be reduced in cows fed species-rich herbage, but plant species offering the best protection against BH are yet to be elucidated. The aim of the present study was to investigate differences in rumen in vitro BH of ALA and LA between single plant species and feeding regimens. Rumen fluid was collected from cows fed either total mixed ration (TMR), species-rich silage (HERB), or grass silage (GRASS). Five single species (alfalfa, birdsfoot trefoil, chicory, English plantain, and salad burnet) and a grass-clover mixture (white clover and ryegrass) were incubated in three replicas up to 30 h and subsequently analyzed for fatty acid content. Michaelis-Menten kinetics was applied for quantifying the BH rate. BH proceeded at the lowest rate in alfalfa and salad burnet (P < 0.005), and independent of species BH rate was lower in HERB and GRASS compared to TMR (P < 0.001).