Microsatellite instability (MSI) analysis of colorectal cancers is clinically useful to identify patients with hereditary nonpolyposis colorectal cancer (HNPCC) caused by germline mutations of mismatch repair genes. MSI status may also predict cancer response/resistance to certain chemotherapies. We evaluated the MSI Analysis System (Promega Corp.; five mononucleotide and two pentanucleotide repeats) and compared the results to the Bethesda panel, which interrogates five microsatellite loci recommended by the 1997 National Cancer Institute-sponsored MSI workshop (three dinucleotide and two mononucleotide repeats). Thirty-four colorectal cancers were analyzed by both assays. The overall concordance between the two assays was 85% (29 of 34). There was complete concordance between the two assays for all of the MSI-high (11 of 11) and microsatellite stable (MSS; 18 of 18) cases. In the 11 MSI-high cases, all 5 of the mononucleotide loci in the MSI Analysis System demonstrated shifted alleles (100% sensitivity), and each shift resulted in products that were smaller in size than the germline alleles. All (5 of 5) of the cases interpreted as MSI-low by the Bethesda assay were interpreted as MSS by the MSI Analysis System. Our results suggest that the MSI Analysis System is generally superior and may help resolve cases of MSI-low into either MSI-high or MSS.