Biohydrogenation of C18 fatty acids in the rumen of cows, from polyunsaturated and monounsaturated to saturated fatty acids, is lower on clover than on grass-based diets, which might result in increased levels of polyunsaturated fatty acids in the milk from clover-based diets affecting its nutritional properties. The effect of forage type on ruminal hydrogenation was investigated by in vitro incubation of feed samples in rumen fluid. Silages of red clover, white clover and perennial ryegrass harvested in spring growth and in third regrowth were used, resulting in six silages. Fatty acid content was analysed after 0, 2, 4, 6, 8 and 24 h of incubation to study the rate of hydrogenation of unsaturated C18 fatty acids. A dynamic mechanistic model was constructed and used to estimate the rate constants (k, h) of the hydrogenation assuming mass action-driven fluxes between the following pools of C18 fatty acids: C18:3 (linolenic acid), C18:2 (linoleic acid), C18:1 (mainly vaccenic acid) and C18:0 (stearic acid) as the end point. For kC18:1,C18:2 the estimated rate constants were 0.0685 (red clover), 0.0706 (white clover) and 0.0868 (ryegrass), and for kC18:1,C18:3 it was 0.0805 (red clover), 0.0765 (white clover) and 0.1022 (ryegrass). Type of forage had a significant effect on kC18:1,C18:2 (P < 0.05) and a tendency to effect kC18:1,C18:3 (P < 0.10), whereas growth had no effect on kC18:1,C18:2 or kC18:1,C18:3 (P > 0.10). Neither forage nor growth significantly affected kC18:0,C18:1, which was estimated to be 0.0504. Similar, but slightly higher, results were observed when calculating the rate of disappearance for linolenic and linoleic acid. This effect persists regardless of the harvest time and may be because of the presence of plant secondary metabolites that are able to inhibit lipolysis, which is required before hydrogenation of polyunsaturated fatty acids can begin.