Abstract In dairy beef calves fed high-concentrate diets even methane emissions may be lesser than calves fed high-roughage diets there is still room to improvement. Substituting palm oil, an ingredient that is questioned by the impact on deforestation of some regions, by polyunsaturated fatty acids (PUFA) may be a good strategy that needs to be tested. Therefore, the aim of this study was to evaluate the rumen in vitro methane gas production by substituting palm oil by different oils rich in polyunsaturated fatty acids in two different concentrates in T1 the main protein source was SBM (8%) and in T2 the main protein source was corn DDG (15%); the remaining concentrate ingredients were 50% corn, 15 to 13% barley, 15 to 16% wheat middlings, beet pulp (5.5% in T1), 1.8% calcium carbonate, 0.5% salt, 0.2% premix. Palm oil was substituted (4 or 3.5 % in T1 or T2, respectively) by canola oil (CO), sunflower oil (SU) or soybean meal oil (SO), so 8 treatments were tested (T1, T1-CO, T1-SU, T1-SO, T2, T2-CO, T2-SU, T2-SO), which are rich in polyunsaturated fatty acids in two. Samples were 1-mm grinded at 1-mm and used in an in vitro digestibility assay as described by [Goeser and Combs (2009)]. The rumen liquid, obtained from rumens at slaughterhouse of beef animals fed high-concentrate diets with a close formula to T1 and T2, was strained through a cheesecloth, collected into pre-warmed thermal flasks, and kept at 39ºC until being used. Two incubation sets were done in parallel and in triplicate: one lasted 48 hours to calculate the DM digestibility; the second lasted 12 days to monitor the methane gas production. A tendency (P = 0.10) in the interaction among basal diet and oil source was observed in total methane production corrected by blank methane production; numerically the same trend (P = 0.14) was observed in net volume of CH4 per degraded DM. After 12 days, the least methane production values were observed in T1-SO and T2-CO (30 and 29 mL ± 4.6, respectively), while the largest methane values were observed in T1-SU (45 ± 4.6 mL) being the remaining treatments intermediate values (42, 39, 31, 38, 41 ± 4.6 mL for T1, T1-CO, T2, T2-SU, T2-SO, respectively). In conclusion, depending on the concentrate formula (protein source used) the effect of fat source used to substitute palm oil and reduce methane emission may differ.