Aims: The aim of this study was to isolate a thermotolerant micro-organism that produces polyhydroxyalkanoates (PHAs) composed of medium-chain-length (mcl) HA units from a biodiesel fuel (BDF) by-product as a carbon source. Methods and Results: We successfully isolated a thermotolerant micro-organism, strain SG4502, capable to accumulate mcl-PHA from a BDF by-product as a carbon source at a cultivation temperature of 45 degrees C. The strain could also produce mcl-PHA from acetate, octanoate and dodecanoate as sole carbon sources at cultivation temperatures up to 55 degrees C. Taxonomic studies and 16S rRNA gene sequence analysis revealed that strain SG4502 was phylogenetically affiliated with species of the genus Pseudomonas. This study is the first report of PHA synthesis by a thermotolerant Pseudomonas. Conclusions: A novel thermotolerant bacterium capable to accumulate mcl-PHA from a BDF by-product was successfully isolated. Significance and Impact of the Study: A major issue regarding industrial production of microbial PHAs is their much higher production cost compared with conventional petrochemical-based plastic materials. Especially significant are the cost of a fermentative substrate and the running cost to maintain a temperature suitable for microbial growth. Thus, strain SG4502, isolated in this study, which assimilates BDF by-product and produces PHA at high temperature, would be very useful for practical application in industry.