Complete relaxation matrix analysis of the off-resonance ROESY (O-ROESY) spectra is presented and demonstrated for a synthetic DNA duplex with two G-A mismatches, d(GCTGTC-GAAAGC)2, in solution. The internuclear distance and the rotational correlation time of the internuclear vector could be garnered simultaneously using complete relaxation matrix analysis of O-ROESY, by which spin diffusion effects could be accommodated. Correlation times in the terminal and the mismatched regions were significantly reduced compared to those in other regions, indicating the conformational flexibility of the mismatched pair. The average structure obtained by restrained molecular dynamics simulation with inclusion of variations of the rotational correlation times also indicated a general tendency of the mismatched and contiguous bases to flip to the outside of the double strand. Off-resonance ROESY combined with the complete relaxation matrix analysis method may offer an alternative way to investigate the structures and dynamics of biological macromolecules.