The DNA mismatch repair (MMR) system is a major DNA repair system that suppresses both inherited and sporadic cancers in humans. In eukaryotes, the MutSα-dependent and MutSβ-dependent MMR pathways correct DNA polymerase errors. Here, we investigated these two pathways on a whole genome level inSaccharomyces cerevisiae. We found that inactivation of MutSα-dependent MMR increases the genome-wide mutation rate by ∼17-fold and loss of MutSβ-dependent MMR elevates the genome-wide mutation rate by ∼4-fold. We also found that MutSα-dependent MMR does not show a preference for protecting coding or noncoding DNA from mutations, whereas MutSβ-dependent MMR preferentially protects noncoding DNA from mutations. The most frequent mutations in themsh6Δstrain are C>T transitions, whereas 1- to 6-bp deletions are the most common genetic alterations in themsh3Δstrain. Strikingly, MutSα-dependent MMR is more important than MutSβ-dependent MMR for protection from 1-bp insertions, while MutSβ-dependent MMR has a more critical role in the defense against 1-bp deletions and 2- to 6-bp indels. We also determined that a mutational signature of yeastMSH6loss is similar to mutational signatures of human MMR deficiency. Furthermore, our analysis showed that compared to other 5′-NCN-3′ trinucleotides, 5′-GCA-3′ trinucleotides are at the highest risk of accumulating C>T transitions at the central position in themsh6Δcells and that the presence of a G/A base at the −1 position is important for the efficient MutSα-dependent suppression of C>T transitions. Our results highlight key differences between the roles of the MutSα-dependent and MutSβ-dependent MMR pathways.