High temperature causes ubiquitous environmental stress to microorganisms, but studies have not fully explained whether and to what extent heat shock would affect genome stability. Hence, this study explored heat-shock-induced genomic alterations in the yeast Saccharomyces cerevisiae. Using genetic screening systems and customized single nucleotide polymorphism (SNP) microarrays, we found that heat shock (52 °C) for several minutes could heighten mitotic recombination by at least one order of magnitude. More than half of heat-shock-induced mitotic recombinations were likely to be initiated by DNA breaks in the S/G2 phase of the cell cycle. Chromosomal aberration, mainly trisomy, was elevated hundreds of times in heat-shock-treated cells than in untreated cells. Distinct chromosomal instability patterns were also observed between heat-treated and carbendazim-treated yeast cells. Finally, we demonstrated that heat shock stimulates fast phenotypic evolutions (such as tolerance to ethanol, vanillin, fluconazole, and tunicamycin) in the yeast population. This study not only provided novel insights into the effect of temperature fluctuations on genomic integrity but also developed a simple protocol to generate an aneuploidy mutant of yeast.