The effect of pasteurisation and co-pasteurisation on biochemical methane potential values in anaerobic digestion (AD) was studied. Pasteurisation prior to digestion in a biogas plant is a common hygienisation method for organic materials which contain or have been in contact with animal by-products. Tests were carried out on food waste, slaughterhouse waste, animal blood, cattle slurry, potato waste, card packaging and the organic fraction of municipal solid waste (OFMSW); pasteurisation at 70 °C for 1 h was applied. Pasteurisation had increased the methane yields of blood (+15%) and potato waste (+12%) only, which both had a low content of structural carbohydrates (hemi-cellulose and cellulose) but a particularly high content of either non-structural carbohydrates such as starch (potato waste) or proteins (blood). With food waste, card packaging and cattle slurry, pasteurisation had no observable impact on the methane yield. Slaughterhouse waste and OFMSW yielded less methane after pasteurisation in the experiments (but statistical significance of the difference between pasteurised and unpasteurised slaughterhouse waste or OFMSW was not confirmed in this work). It is concluded that pasteurisation can positively impact the methane yield of some specific substrates, such as potato waste, where heat-treatment may induce gelatinisation with release of the starch molecules. For most substrates, however, pasteurisation at 70 °C is unlikely to increase the methane yield. It is unlikely to improve biodegradability of lignified materials, and it may reduce the methane yield from substrates which contain high contents of volatile components. Furthermore, in this experimental study, the obtained methane yield was unaffected by whether the substrates were pasteurised individually and then co-digested or co-pasteurised as a mixture before batch digestion.