Next-generation sequencing (NGS) panels are widely used for defining tumor mutation profiles and determining treatment approaches. We performed targeted NGS with 382 colorectal cancer genes with known microsatellite instability (MSI). After exclusion of germline alterations, the load of somatic mutations and small insertion/deletion (indel) alterations were determined. In the test set, 79 patients with 41 microsatellite-stable (MSS) and 38 MSI tumors were included. There were 120 MSS and eight MSI-high tumors in the validation set. The number of somatic mutations of whole samples were distinguished into three groups: mutant functional polymerase epsilon catalytic subunit, MSI, and MSS tumors. The median numbers of somatic and indel mutations in MSI tumors were higher. The indel mutation to whole mutation ratio (I index) was higher in MSI tumors. Hypermutation and low I index of polymerase epsilon catalytic subunit mutant tumors, a somatic mutation load cut-off of ≥40, and an I index of ≥9% were selected as the criteria for detecting MSI tumors with high sensitivity and specificity. With the analysis of alteration patterns of homopolymer genes, a higher median number of homopolymer mutations in MSI tumors was observed. Mutated homopolymer ≥5 was selected as the criterion for detecting MSI tumors. MSI in colorectal cancer can be detected by targeted NGS panels with high sensitivity and specificity using somatic mutation load and I index.