The functional characterization of nonsynonymous single nucleotide polymorphisms in human mismatch repair (MMR) genes has been critical to evaluate their pathogenicity for hereditary nonpolyposis colorectal cancer. We previously established an assay for detecting loss-of-function mutations in the MLH1 gene using a dominant mutator effect of human MLH1 expressed in Saccharomyces cerevisiae. The purpose of this study is to extend the functional analyses of nonsynonymous single nucleotide polymorphisms in the MLH1 gene both in quality and in quantity, and integrate the results to evaluate the variants for pathogenic significance. The 101 MLH1 variants, which covered most of the reported MLH1 nonsynonymous single nucleotide polymorphisms and consisted of one 3-bp deletion, 1 nonsense and 99 missense variants, were examined for the dominant mutator effect by three yeast assays and for the ability of the variant to repair a heteroduplex DNA with mismatch bases by in vitro MMR assay. There was diversity in the dominant mutator effects and the in vitro MMR activities among the variants. The majority of functionally inactive variants were located around the putative ATP-binding pocket of the NH2-terminal domain or the whole region of the COOH-terminal domain. Integrated functional evaluations contribute to a better prediction of the cancer risk in individuals or families carrying MLH1 variants and provide insights into the function-structure relationships in MLH1.