Microsatellite instability (MSI) serves as an important therapeutic and prognostic marker. Immunohistochemistry (IHC) and polymerase chain reaction (PCR) have long been regarded as the "gold standard" for MSI detection. In recent years, next-generation sequencing (NGS)-based methods, offering expanded target coverage of microsatellite (MS) loci and improved analytical performance, have gained widespread acceptance. However, discrepancies between NGS and traditional methods have been occasionally reported. In this study, we conducted a large-scale retrospective analysis of MSI results of 35,563 Chinese pan-cancer cases which underwent a NGS test in a central clinical laboratory. Our work introduced a novel algorithm for NGS-based MSI detection, examined MSI-H prevalence and value distribution across cancer types, identifies MSI-associated genes and variants, evaluates the MSI detection discordance between PCR and NGS in a pan-cancer context, and distilled 7 MS loci suitable for pan-cancer MSI detection.