This research presents a method for the positive selection of mutants with improved xylose and L-arabinose fermentation in the thermotolerant, naturally xylose-utilizing yeast Ogataea polymorpha which is based on isolation of the mutants growing on L-arabinose as sole carbon and energy source. Whole-genome sequencing of the most efficient xylose-fermenting strain, A107, revealed mutations in the API1 and IRA1 genes, which are homologous to bacterial arabinose-5-phosphate isomerase and the Ras-GTPase activating domain in Saccharomyces cerevisiae, respectively. Disruption of the IRA1 gene increased ethanol production during the fermentation of xylose and L-arabinose in O. polymorpha at 45 °C. Overexpression of the API1 gene specifically enhanced L-arabinose fermentation without affecting xylose fermentation. The most productive mutant strain accumulated 20.91 g/L of ethanol in a xylose-containing medium at 45 °C, exceeding the ethanol accumulation level of the wild-type strain (0.40 g/L) by over 50 times. This strain holds potential for application in simultaneous saccharification and fermentation (SSF) processes.