High-temperature fermentation using thermophilic microorganisms may provide cost-effective processes for the industrial production of fuels and chemicals, due to decreased hygiene and cooling costs. In the present study, the genetically trackable thermophile Parageobacillus thermoglucosidasius DSM2542(T) was engineered to produce (2R, 3R)-butanediol (R-BDO), a valuable chemical with broad industrial applications. The R-BDO biosynthetic pathway was optimized by testing different combinations of pathway enzymes, with acetolactate synthase (AlsS) from Bacillus subtilis and acetolactate decarboxylase (AlsD) from Streptococcus thermophilus yielding the highest production in P. thermoglucosidasius DSM2542(T). Following fermentation condition optimization, shake flask fermentation at 55 degrees C resulted in the production of 7.2 g/L R-BDO with similar to 72% theoretical yield. This study details the microbial production of R-BDO at the highest fermentation temperature reported to date and demonstrates that P. thermoglucosidasius DSM2542(T) is a promising cell factory for the production of fuels and chemicals using high-temperature fermentation.