The accurate identification and interpretation of germline mutations in mismatch repair genes in colorectal cancer cases is critical for clinical management. Current data suggest that mismatch repair mutations are highly heterogeneous and that many mutations are not detected when conventional DNA sequencing alone is used. OBJECTIVE: To evaluate the potential of conversion analysis compared with DNA sequencing alone to detect heterogeneous germline mutations in MLH1, MSH2, and MSH6 in colorectal cancer patients. DESIGN, SETTING, AND PARTICIPANTS: Multicenter study with patients who participate in the Colon Cancer Family Registry. Mutation analyses were performed in participant samples determined to have a high probability of carrying mismatch repair germline mutations. Samples from a total of 64 hereditary nonpolyposis colorectal cancer cases, 8 hereditary nonpolyposis colorectal cancer-like cases, and 17 cases diagnosed prior to age 50 years were analyzed from June 2002 to June 2003. MAIN OUTCOME MEASURES: Classification of family members as carriers or noncarriers of germline mutations in MLH1, MSH2, or MSH6; mutation data from conversion analysis compared with genomic DNA sequencing. RESULTS: Genomic DNA sequencing identified 28 likely deleterious exon mutations, 4 in-frame deletion mutations, 16 missense changes, and 22 putative splice site mutations. Conversion analysis identified all mutations detected by genomic DNA sequencing--plus an additional exon mutation, 12 large genomic deletions, and 1 exon duplication mutation--yielding an increase of 33% (14/42) in diagnostic yield of deleterious mutations. Conversion analysis also showed that 4 of 16 missense changes resulted in exon skipping in transcripts and that 17 of 22 putative splice site mutations affected splicing or mRNA transcript stability. Conversion analysis provided an increase of 56% (35/63) in the diagnostic yield of genetic testing compared with genomic DNA sequencing alone.