Microsatellite instability (MSI) has emerged as a predictive biomarker for immunotherapy response in various cancers, but its role in non-small cell lung cancer (NSCLC) is not fully understood.The authors used the bioinformatics tool MIAmS to assess microsatellite status from next-generation sequencing (NGS) data using a tailored microsatellite score. Immunohistochemistry (IHC) assays were also performed to evaluate the correspondence between MSI and deficient mismatch repair (dMMR) status. A retrospective analysis of 1547 lung cancer patients was conducted, focusing on those with an MSI phenotype. Clinical characteristics, co-occurring molecular alterations, tumor mutation burden (TMB), and homologous recombination deficiency (HRD) status were evaluated in this subset.Of the 1547 patients analyzed, eight (0.52%) were identified as having MSI through MIAmS, with six (0.39%) of these cases also being dMMR on IHC. All patients with dMMR had an MS score ≥2 and a history of smoking. Most patients showed loss of MLH1 and PMS2 staining on IHC. No correlation was found between MSI status and programmed death-ligand 1 expression, although all MSI patients exhibited high TMB, averaging 21.4 ± 5.6 mutations per megabase.MSI/dMMR in lung cancer is exceedingly rare, affecting less than 1% of cases. NGS-based analysis combined with bioinformatics tools provides a robust method to identify MSI/dMMR patients, potentially guiding immunotherapy decisions. This comprehensive approach integrates molecular genotyping and MSI detection, offering personalized treatment options for lung cancer patients. NGS-based MSI testing is emerging as the preferred method for detecting microsatellite instability in various tumor types, including rare cancers.