Instability under non-native processing conditions, especially at elevated temperatures, is a major factor preventing widespread adoption of biocatalysts for industrial synthesis. Glucose dehydrogenase (GDH) is an important cofactor-regeneration system that allows both NAD+ and NADP+ to be regenerated back to NADH and NADPH, respectively. However, the system often suffers from instability at high temperature, low salt, and presence of organic solvents. First, we test the generation of a thermostable GDH via structure-guided consensus. The consensus sequence in combination with additional knowledge-based criteria was used to select amino acids for substitutions. Using this approach we generated 24 variants, 11 of which showed higher thermal stability than the wild-type GDH, a success rate of 46%. Of the 24 variants, the mutations of seven where located at the subunit interface—known to influence GDH stability—and six where more stable (86% success). The best variants feature a half-life of ~ 4 days at 65oC in contrast to ~ 20 minutes at 25oC for the wild type, enhancing stability 106-fold. In addition, the three most stabilizing single mutations were transferred to two GDH homologs from Bacillus thuringiensis and Bacillus licheniformis. The resulting stability changes provides further support that these residues are critical for stability of GDHs and reinforces the success of the consensus approach for identifying stabilizing mutations.