Microsatellite instability (MSI) is a mutator phenotype caused by deficient mismatch repair (dMMR), and high-frequency MSI (MSI-H)/dMMR is increasingly used as a biomarker for immune checkpoint inhibitor (ICI) eligibility in advanced cancers. Next-generation sequencing (NGS) enables comprehensive assessment of MSI status and genomic alterations in a single test. Liquid biopsy-based NGS assays can also determine MSI status, and MSI-H is found in various tumor types, leading to greater adoption of immunotherapy. NGS studies have characterized MSI-driven carcinogenesis, including high rates of fusion kinases in MSI-H colorectal cancers (CRCs), which are targets for kinase inhibitors. NTRK fusions are linked to the colorectal serrated neoplasia pathway. Recent advances in MSI-H research have led to novel diagnostic and therapeutic techniques, such as synthetic lethal therapy targeting the Werner gene. DNA sensing in cancer cells is essential for antitumor immunity induced by dMMR, opening new avenues for immunotherapy. Clinical relevance exists for analyzing MSI and MSI-H-associated genomic alterations in malignancies. This review provides an update on MSI-driven carcinogenesis, emphasizing unique diagnostic and immunotherapeutic strategies. MSI-H is associated with increased mutational burden and neoantigens, making it a promising target for immunotherapy. ARID1A deficiency is linked to CIMP and EBV infection in various cancers. MSI-H CRCs with wild-type KRAS/BRAF often have NTRK fusions. Werner helicase is a synthetic lethal target in MSI-H cancers. The accumulation of gene mutations in MSI-H CRCs leads to decreased HLA-ABC gene expression, contributing to immune evasion. B2M mutations in MSI-H CRCs do not predict primary ICI resistance. The common frameshift mutation landscape in MSI-H cancers supports immunoediting during tumor progression. DNA sensing in dMMR tumor cells is crucial for antitumor immunity. Immune checkpoint inhibitors show promise in MSI-H cancers, with pembrolizumab approved as first-line treatment for MSI-H/dMMR metastatic CRC. Research on MSI-H pathogenesis is advancing diagnostic, therapeutic, and preventive strategies in clinical settings.Microsatellite instability (MSI) is a mutator phenotype caused by deficient mismatch repair (dMMR), and high-frequency MSI (MSI-H)/dMMR is increasingly used as a biomarker for immune checkpoint inhibitor (ICI) eligibility in advanced cancers. Next-generation sequencing (NGS) enables comprehensive assessment of MSI status and genomic alterations in a single test. Liquid biopsy-based NGS assays can also determine MSI status, and MSI-H is found in various tumor types, leading to greater adoption of immunotherapy. NGS studies have characterized MSI-driven carcinogenesis, including high rates of fusion kinases in MSI-H colorectal cancers (CRCs), which are targets for kinase inhibitors. NTRK fusions are linked to the colorectal serrated neoplasia pathway. Recent advances in MSI-H research have led to novel diagnostic and therapeutic techniques, such as synthetic lethal therapy targeting the Werner gene. DNA sensing in cancer cells is essential for antitumor immunity induced by dMMR, opening new avenues for immunotherapy. Clinical relevance exists for analyzing MSI and MSI-H-associated genomic alterations in malignancies. This review provides an update on MSI-driven carcinogenesis, emphasizing unique diagnostic and immunotherapeutic strategies. MSI-H is associated with increased mutational burden and neoantigens, making it a promising target for immunotherapy. ARID1A deficiency is linked to CIMP and EBV infection in various cancers. MSI-H CRCs with wild-type KRAS/BRAF often have NTRK fusions. Werner helicase is a synthetic lethal target in MSI-H cancers. The accumulation of gene mutations in MSI-H CRCs leads to decreased HLA-ABC gene expression, contributing to immune evasion. B2M mutations in MSI-H CRCs do not predict primary ICI resistance. The common frameshift mutation landscape in MSI-H cancers supports immunoediting during tumor progression. DNA sensing in dMMR tumor cells is crucial for antitumor immunity. Immune checkpoint inhibitors show promise in MSI-H cancers, with pembrolizumab approved as first-line treatment for MSI-H/dMMR metastatic CRC. Research on MSI-H pathogenesis is advancing diagnostic, therapeutic, and preventive strategies in clinical settings.