The yeast Saccharomyces cerevisiae has been widely used as a model system for studying the physiological and pharmacological action of small-molecular drugs. Here, a heterozygous diploid S. cerevisiae strain QSS4 was generated to determine whether drugs could induce chromosomal instability by determining the frequency of mitotic recombination. Using the combination of a custom SNP microarray and yeast screening system, the patterns of chromosomal instability induced by drugs were explored at the whole genome level in QSS4. We found that Zeocin (a member of the bleomycin family) treatment increased the rate of genomic alterations, including aneuploidy, loss of heterozygosity (LOH), and chromosomal rearrangement over a hundred-fold. Most recombination events are likely to be initiated by DNA double-stand breaks directly generated by Zeocin. Another remarkable finding is that G4-motifs and low GC regions were significantly underrepresented within the gene conversion tracts of Zeocin-induced LOH events, indicating that certain DNA regions are less preferred Zeocin-binding sites in vivo. This study provides a novel paradigm for evaluating genetic toxicity of small-molecular drugs using yeast models.