The interest in biogas production in Poland is growing rapidly. This is mostly due to the fact that there is a need for handling and managing the increasing quantities of diverse bio-waste generated by industry and agriculture. Therefore, good laboratory practices and correct preparation of batch tests are very important for planning of a full-scale biogas plant. The aim of the paper was to determine the effect of mixing in the laboratory batch reactors on the biogas yield of maize straw under thermophilic conditions. The scope of this work included: (1) the analysis of basic physical and chemical parameters and (2) laboratory determination of biogas and methane yield from anaerobic digestion of maize straw with different frequencies of mixing. The obtained biogas and methane yield from the thermophilic fermentation of maize straw mixed every day was 381.89 m3•Mg-1 FM and 184.97 m3•Mg-1 FM, respectively. The results of this study confirmed the effect of no mixing inside reactors. In the batch test a decrease in biogas and methane yields was observed, by approx. 60 m3•Mg-1 and approx. 28 m3•Mg-1, respectively.