Missense mutations in MLH1 have frequently been detected in patients with Lynch syndrome, but their genetic significance has not been extensively assessed. In this study, we attempt to evaluate the etiological role of eight MLH1 missense variants. The variants were analyzed for their ability to affect MLH1 protein interaction with its partner PMS2 in vivo employing a yeast two-hybrid system. In addition, a SIFT (sorting intolerant from tolerant) algorithm was adopted to predict the effects of amino acid substitutions. Finally, scanning of mutations in a normal Chinese population and assay of the clinical characteristics have all been taken into account. Our results demonstrated that the MLH1 variants D485E and L653R cause functional alterations of the human MutLα complex significantly. The R265C, D304V, A586P, and R755S variants affect partial interaction. The remaining two variants, N38D and L559R, could be nonfunctional polymorphisms or might affect the mismatch repair system through other mechanisms.