The aim of the study was to assess impacts of increasing dietary levels of glycerol on in vitro ruminal fermentation and CH₄ production from a barley based feedlot diet. Glycerol was used as replacement for barley grain at inclusions of 0, 70, 140 and 210g/kg of diet dry matter (DM) in a diet containing an equal mixture of barley grain and barley silage. Both grain and silage were dried and ground through a 1mm screen before mixing with glycerol. The experiment was repeated (n =2) using ANKOM® bags in 50ml sealed batch culture serum vials (i.e., 0.5g substrate+25ml media) with a 3:1 ratio of buffer:rumen liquor (n =5 bags/treatment/experiment). Rumen liquor was obtained from two cows fed a diet containing 710g/kg barley silage, 250g/kg barley grain and 40g/kg concentrate (DM basis). Gas production was measured by water displacement at 3, 6, 12, 24, 36 and 48h after inoculation. Volumes corrected for gas released from 15 negative controls (i.e., no substrate) were used to estimate net gas production at 24 and 48h. Gas samples collected at 24 and 48h were analyzed for CH₄ concentration. In vitro DM disappearance (IVDMD) and culture pH were measured at 48h. Cumulative gas production as ml/g DM substrate and IVDMD were similar among treatments. Culture pH did not differ among treatments. Total CH₄ production (as mg or as mg CH₄/g digested DM) did not differ among treatments. Results suggest that replacing barley grain with glycerol did not reduce in vitro CH₄ production as a function of digested DM or substrate DM. This article is part of the special issue entitled: Greenhouse Gases in Animal Agriculture - Finding a Balance between Food and Emissions, Guest Edited by T.A. McAllister, Section Guest Editors; K.A. Beauchemin, X. Hao, S. McGinn and Editor for Animal Feed Science and Technology, P.H. Robinson.