In eukaryotes, the recognition of the DNA postreplication errors and initiation of the mismatch repair is carried out by two MutS homologs: MutSα and MutSβ. MutSα recognizes base mismatches and 1 to 2 unpaired nucleotides whereas MutSβ recognizes longer insertion-deletion loops (IDLs) with 1 to 15 unpaired nucleotides as well as certain mismatches. Results from molecular dynamics simulations of native MutSβ:IDL-containing DNA and MutSα:mismatch DNA complexes as well as complexes with swapped DNA substrates provide mechanistic insight into how the differential substrate specificities are achieved by MutSα and MutSβ, respectively. Our simulations results suggest more extensive interactions between MutSβ and IDL-DNA and between MutSα and mismatch-containing DNA that suggest corresponding differences in stability. Furthermore, our simulations suggest more expanded mechanistic details involving a different degree of bending when DNA is bound to either MutSα or MutSβ and a more likely opening of the clamp domains when noncognate substrates are bound. The simulation results also provide detailed information on key residues in MutSβ and MutSα that are likely involved in recognizing IDL-DNA and mismatch-containing DNA, respectively.