Abstract: This study set out to assess the impact of a total mixed ration (TMR) based on lucerne straw on methane production and in vitro digestibility. Different TMRs were created using concentrates and wheat straw in a 30:70 ratio, which served as the substrate for in vitro study. As a control, the TMR without lucerne straw was assigned the label L0. The study examined the in vitro DMD and methane production of TMRs that replaced 10%, 20%, 30%, 40%, and 50% of the wheat straw with lucerne straw. These TMRs were designed as L1, L2, L3, L4, L5, and L6, respectively. Gas chromatography was used to estimate the production of methane. When lucerne straw was included in TMRs at all levels, in vitro experiments showed an improvement in dry matter digestibility when compared to the control group. However, adding 35% lucerne straw to TMR instead of 35% wheat straw resulted in a significant (p<0.05) increase in in vitro DM digestibility (9.70%). Similar to the control group, all lucerne straw-based TMRs showed a decrease in in vitro methane generation. Compared to the control group, which produced 2.34 ml/100 mg DM, in vitro methane production was 20.94% lower in TMR containing 35% lucerne straw (1.85 ml/100 mg DM). Compared to the control group, in vitro methane production dropped by 27.76% in terms of ml/100 mg DMD at a 35% inclusion level of lucerne straw. Therefore, adding 50% (w/w) more lucerne straw to 70% of the wheat straw in a 70:30 TMR blend increases DMD and decreases methane synthesis in vitro.