The article provides an updated overview of microsatellite instability (MSI) and its implications in cancer diagnosis and treatment. MSI, resulting from deficient mismatch repair (dMMR), is a prominent mutator phenotype that has gained increasing interest as a biomarker for advanced cancer patients to determine their eligibility for immune checkpoint inhibitors (ICIs). Next-generation sequencing (NGS) methods, including comprehensive genomic profiling (CGP) and liquid biopsy-based assays, have been developed to accurately assess MSI status. MSI-H has been identified in various tumor types, leading to the broader adoption of immunotherapy. NGS studies have characterized MSI-driven carcinogenesis, highlighting significant rates of fusion kinases in colorectal cancers (CRCs) with MSI-H, particularly in MLH1-methylated CRCs with wild-type KRAS/BRAF. Recent advances include the development of novel diagnostic and therapeutic techniques, such as synthetic lethal therapy targeting the Werner gene. DNA sensing in cancer cells is crucial for antitumor immunity induced by dMMR, opening new avenues and biomarkers for immunotherapy. The article emphasizes unique landscapes of diagnostic and immunotherapeutic strategies for MSI-driven carcinogenesis.The article provides an updated overview of microsatellite instability (MSI) and its implications in cancer diagnosis and treatment. MSI, resulting from deficient mismatch repair (dMMR), is a prominent mutator phenotype that has gained increasing interest as a biomarker for advanced cancer patients to determine their eligibility for immune checkpoint inhibitors (ICIs). Next-generation sequencing (NGS) methods, including comprehensive genomic profiling (CGP) and liquid biopsy-based assays, have been developed to accurately assess MSI status. MSI-H has been identified in various tumor types, leading to the broader adoption of immunotherapy. NGS studies have characterized MSI-driven carcinogenesis, highlighting significant rates of fusion kinases in colorectal cancers (CRCs) with MSI-H, particularly in MLH1-methylated CRCs with wild-type KRAS/BRAF. Recent advances include the development of novel diagnostic and therapeutic techniques, such as synthetic lethal therapy targeting the Werner gene. DNA sensing in cancer cells is crucial for antitumor immunity induced by dMMR, opening new avenues and biomarkers for immunotherapy. The article emphasizes unique landscapes of diagnostic and immunotherapeutic strategies for MSI-driven carcinogenesis.