Molecular dynamics (MD) simulations were conducted for a G-T mismatch-containing DNA decamer, d(CCATGCGTGG)(2), and its Watson-Crick parent sequence, d(CCACGCGTGG)(2). Dynamics in unrestrained MD trajectories were in poor agreement with prior C-13 NMR studies. However, the accuracy of the trajectories was improved by the use of time-averaged interatomic distance restraints derived from H-1 NMR. Postprocess smoothing of the trajectories further improved accuracy. Comparison of restrained and smoothed trajectories of the two DNA molecules revealed distinct differences in dynamics. The major groove width of the mismatched oligomer was more variable over the course of the simulation compared to its parent sequence. Greater variability in helical parameters stretch and opening for the mismatches indicated less kinetically stable base pairing. Interbase helical parameters rise, roll, and tilt were also more variable in certain base steps involving mismatched bases. These dynamic differences between normal and G-T mismatched DNA reflect differences in local flexibility that may play a role in mismatch recognition by the MutS. A potential alternate G-T mismatch binding mode for MutS is also proposed.