Abstract: The genetic basis of organisms' adaptation to different environments is a central issue of molecular evolution. The budding yeast Saccharomyces cerevisiae and its relatives predominantly ferment glucose into ethanol even in the presence of oxygen. This was suggested to be an adaptation to glucose-rich habitats, but the underlying genetic basis of the evolution of aerobic fermentation remains unclear. In S. cerevisiae, the first step of glucose metabolism is transporting glucose across the plasma membrane, which is carried out by hexose transporter (Hxt) proteins. Although several studies have recognized that the rate of glucose uptake can affect how glucose is metabolized, the role of HXT genes in the evolution of aerobic fermentation has not been fully explored. In this study, we identified all members of the HXT gene family in 23 fully sequenced fungal genomes, reconstructed their evolutionary history to pinpoint gene gain and loss events and evaluated their adaptive significance in the evolution of aerobic fermentation. We found that the HXT genes have been extensively amplified in the two fungal lineages that have independently evolved aerobic fermentation. In contrast, reduction of the number of HXT genes has occurred in aerobic respiratory species. Our study reveals a strong positive correlation between the copy number of HXT genes and the strength of aerobic fermentation, suggesting that HXT gene expansion has facilitated the evolution of aerobic fermentation.