Changing thermal environments drives the evolution of populations. To clarify the evolutionary process of thermal tolerance acquisition in teleosts (i.e., new mutation, introgression, and standing genetic variation), we examined the genomic and phenotypic features of the Japanese fluvial sculpin Cottus pollux as a good model. This species includes two migratory patterns; F type is fluvial and vulnerable to high temperature, whereas A type is amphidromous and less vulnerable to such condition. In western Japan, several F type populations show discordance between the migration type and mtDNA type possessing the A type mtDNA. We focused on one of such population in the Minami River (Fukui Pref.) and examined the origin of the “discordant” population and molecular basis of high-temperature adaptation by gene tree analysis using genome-wide transcriptome sequences. Further, phenotypic variation in high-temperature tolerance was quantified. Our results indicated the hybrid origin for the “discordant” population. We also found that the “discordant” population and the A type population shared alleles of the major thermal tolerance loci, which were likely obtained through introgression from the A type. These results suggest that the introgression of such loci facilitated colonization of the fluvial sculpin to high-temperature environments.