Background Environmental stresses and inhibitors encountered by Saccharomyces cerevisiae strains are the main limiting factors in bioethanol fermentation. Strains with different genetic backgrounds usually show diverse stress tolerance responses. An understanding of the mechanisms underlying these phenotypic diversities within S. cerevisiae populations could guide the construction of strains with desired traits. Results We explored the genetic characteristics of the bioethanol S. cerevisiae strain YJS329 and elucidated how genetic variations in its genome were correlated with specified traits compared to similar traits in the S288c-derived strain, BYZ1. Karyotypic electrophoresis combined with array-comparative genomic hybridization indicated that YJS329 was a diploid strain with a relatively constant genome as a result of the fewer Ty elements and lack of structural polymorphisms between homologous chromosomes that it contained. By comparing the sequence with the S288c genome, a total of 64,998 SNPs, 7,093 indels and 11 unique genes were identified in the genome of YJS329-derived haploid strain YJSH1 through whole-genome sequencing. Transcription comparison using RNA-Seq identified which of the differentially expressed genes were the main contributors to the phenotypic differences between YJS329 and BYZ1. By combining the results obtained from the genome sequences and the transcriptions, we predicted how the SNPs, indels and chromosomal copy number variations may affect the mRNA expression profiles and phenotypes of the yeast strains. Furthermore, some genetic breeding strategies to improve the adaptabilities of YJS329 were designed and experimentally verified. Conclusions Through comparative functional genomic analysis, we have provided some insights into the mechanisms underlying the specific traits of the bioenthanol strain YJS329. The work reported here has not only enriched the available genetic resources of yeast but has also indicated how functional genomic studies can be used to improve genetic breeding in yeast.