Background Colorectal tumors caused by failure of the DNA mismatch repair system commonly show microsatellite instability. Our goals were to compare the performance of two panels of markers(a panel previously recommended by the National Cancer Institute[NCI] and a pentaplex of mononucleotide repeats) and to devise the simplest diagnostic strategy for identification of patients with colorectal cancer characterized by defects in mismatch repair. Methods We recruited 1058 patients who were newly diagnosed with colorectal cancer. DNA from fresh-frozen and paraffin-embedded tumors was tested for microsatellite instability, using the NCI-recommended panel of microsatellite markers and the pentaplex panel of mononucleotide repeats, respectively, as templates for polymerase chain reactions(PCRs). Microsatellite instability in fresh-frozen tumors was also assessed using the pentaplex panel of mononucleotides in a crossover analysis. The expression of mismatch repair proteins(MLH1, MSH2, MSH6, and PMS2) in the tumors was determined immunohistochemically. The sensitivity and specificity with which the marker panels identified tumors with deficiencies in the expression of mismatch repair proteins were calculated. All statistical tests were two-sided. Results The sensitivity and positive predictive value of the NCI panel were 76.5%(95% confidence interval[CI] = 61% to 92%) and 65.0%(95% CI = 49% to 81%), respectively; corresponding values for the mononucleotide pentaplex panel were 95.8%(95% CI = 89% to 103%) and 88.5%(95% CI = 79% to 98%), respectively. A panel consisting of the mononucleotide repeat markers BAT26 and NR24 alone had the same predictive value as the pentaplex panel of mononucleotide repeats. Conclusions. The pentaplex panel of mononucleotide repeats performs better than the NCI panel for the detection of mismatch repair-deficient tumors. Simultaneous assessment of the instability of BAT26 and NR24 is as effective as use of the pentaplex panel for diagnosing mismatch repair deficiency.