Identification of germline mutations in DNA mismatch repair genes in colorectal cancer probands without an extensive family history can be problematic when ascribing relevance to cancer causation. We undertook a structured assessment of the disease-causing potential of sequence variants identified in a prospective, population-based study of 932 colorectal cancer patients, diagnosed at <55 years of age. Patient samples were screened for germline mutations in MLH1, MSH2, and MSH6. Of 110 carriers, 74 (67%) had one of 33 rare variants of uncertain pathogenicity (12 MLH1, 11 MSH2, and 10 MSH6). Pathogenicity was assessed by determining segregation in families, allele frequency in large numbers of unaffected controls, effect on mRNA for putative splice-site mutations, effect on protein function by bioinformatic analysis and tumor microsatellite instability (MSI) status and DNA mismatch repair protein expression by immunohistochemistry. Because of the ambiguous nature of these variants and lack of concordance between functional assays and control allele frequency, we devised a scoring system to rank the degree of support for a pathogenic role. MLH1 c.200G>A p.G67E, MLH1 c.2041G>A p.A681T, and MSH2 c.2634+5G>C were categorized as pathogenic through assimilation of all available data, while 14 variants were categorized as benign (seven MLH1, three MSH2, and four MSH6). Interestingly, there is tentative evidence suggesting a possible protective effect of three variants (MLH1 c.2066A>G pQ689R, c.2146G>A p.V716M, and MSH2 c.965G>A p.G322D). These findings support a causal link with colorectal cancer for several DNA mismatch repair gene variants. However, the majority of missense changes are likely to be inconsequential polymorphisms.