The yeast species Hyphopichia is common in nature and strongly competitive under harsh environmental conditions. Here, we characterized Hyphopichia burtonii KJJ43 and H. pseudoburtonii KJS14, which exhibit strong halotolerance, using genomic and transcriptomic analyses. The genomes of H. burtonii and H. pseudoburtonii comprised eight chromosomes with 85.17% nucleotide identity and significant divergence in synteny. Notably, both Hyphopichia genomes possessed extended gene families of amino acid permeases and ATP-binding cassette (ABC) transporters, whose dynamic expression patterns during osmotic stress were revealed using transcriptome profiling. Intriguingly, we found unique features of the HOG pathway activated by Hog1p even under non-osmotic stress conditions and the upregulation of cytosolic Gpd1 protein during osmotic stress. Associated with hyperfilamentation growth under high osmotic conditions, a set of genes in the FLO family with induced expression in response to NaCl, KCl, and sorbitol supplementation were identified. Moreover, comparative transcriptome analysis reveals the NaCl-specific induction of genes involved in amino acid biosynthesis and metabolism, particularly BAT2. This suggests the potential association between oxoacid reaction involving branched-chain amino acids and osmotolerance. The combined omics analysis of two Hyphopichia species provides insights into the novel mechanisms involved in salt and osmo-stress tolerance exploited by diverse eukaryotic organisms.