Distillation of fermented sugarcane juice produces both rum and cachaça, significant sources of revenue in Brazil and elsewhere. In this study, we provide a genomic analysis of a Saccharomyces cerevisiae strain isolated from a cachaça distillery in Brazil. We determined the complete genome sequence of a strain with high flocculation capacity, high tolerance to ethanol, osmotic and heat shock stress and high fermentation rates and compared the sequence with that of the reference S288c genome as well as those of two other cachaça strains. Single-nucleotide polymorphism analysis identified alterations in genes involved in nitrogen and organic compound metabolism, integrity of organelles and ion homeostasis. The strain exhibited fragmentation of several flocculation genes relative to the reference genome, as well as loss of a stop codon in the FLO8 gene, which encodes a transcription factor required for FLO gene expression. The strain contained no genes not present in the reference genome strain but did lack several genes, including asparaginase genes, maltose utilization loci, and several genes from the tandem array of the DUP240 family. The three cachaça strains lacked different sets of genes, but the asparaginase genes and several of the DUP240 genes were common deficiencies. This study provides new insights regarding the selective pressure of sugarcane fermentation on the genome of yeast strains and offers additional genetic resources for modern synthetic biology and genome editing tools., (© 2021. Sociedade Brasileira de Microbiologia.)