The industrial yeast Saccharomyces cerevisiae has a plastic genome with a great flexibility in adaptation to varied conditions of nutrition, temperature, chemistry, osmolarity, and pH in diversified applications. A tolerant strain against 2-furaldehyde (furfural) and 5-hydroxymethyl-2-furaldehyde (HMF) was successfully obtained previously by adaptation through environmental engineering toward development of the next-generation biocatalyst. Using a time-course comparative transcriptome analysis in response to a synergistic challenge of furfural-HMF, here we report tolerance phenotypes of pathway-based transcriptional profiles as components of the adapted defensive system for the tolerant strain NRRL Y-50049. The newly identified tolerance phenotypes were involved in biosynthesis superpathway of sulfur amino acids, defensive reduction-oxidation reaction process, cell wall response, and endogenous and exogenous cellular detoxification. Key transcription factors closely related to these pathway-based components, such as Yap1, Met4, Met31/32, Msn2/4, and Pdr1/3, were also presented. Many important genes in Y-50049 acquired an enhanced transcription background and showed continued increased expressions during the entire lag phase against furfural-HMF. Such signature expressions distinguished tolerance phenotypes of Y-50049 from the innate stress response of its progenitor NRRL Y-12632, an industrial type strain. The acquired yeast tolerance is believed to be evolved in various mechanisms at the genomic level. Identification of legitimate tolerance phenotypes provides a basis for continued investigations on pathway interactions and dissection of mechanisms of yeast tolerance and adaptation at the genomic level.