Germline mutations in the mismatch repair genes mutL homolog 1 (MLH1) and mutS homolog 2 (MSH2), MSH6, and postmeiotic segregation increased 2 (PMS2) lead to the development of hereditary nonpolyposis colorectal cancer (HNPCC). Diagnosis of HNPCC relies on the compilation of a thorough family history of cancer, documentation of pathological findings, tumor testing for microsatellite instability (MSI) and immunohistochemistry (IHC), and germline mutation analysis of the suspected genes. As a hallmark of HNPCC, microsatellite instability is widely accepted as a primary method for identifying individuals at risk for HNPCC. It serves as an excellent, easy-to-evaluate marker of mismatch repair deficiency. Recent improvements in MSI testing have significantly enhanced the accuracy and reduced its cost. Proficiency testing for MSI is available, and laboratory-to-laboratory reproducibility of such testing can be easily evaluated. In addition, the combination of microsatellite instability testing, MLH1 promoter methylation analysis, and BRAF (V600E) mutation analysis can distinguish a sporadic colorectal cancer from one associated with HNPCC, helping to avoid costly molecular genetic testing for germline mutations in mismatch repair genes. In this article, we discuss the development of MSI markers used for HNPCC screening and focus on the advantages and disadvantages of MSI testing in screening for HNPCC patients. We conclude that MSI is as sensitive and specific as IHC, given its excellent reproducibility and its potential capability to indicate mutations not be detected by IHC. MSI has been used and will continue to prevail as the primary screening tool for identifying HNPCC patients.