Background: Lynch Syndrome (LS), known as hereditary nonpolyposis colorectal cancer (HNPCC1), is caused by germline mutations in the mismatch repair (MMR) genes (MLH1, MSH2, PMS2, MSH6), or epigenetic silencing of MSH2 which caused by deletion of 3-prime exons of the EPCAM gene and intergenic regions directly upstream of the MSH2 gene. DNA mismatches commonly occur in regions of repetitive nucleotide sequences called microsatellites, and the status of microsatellite instability (MSI) is the key feature in LS-associated cancers. Although there are sporadic reports of MSI and LS in certain cohorts, the real-world status of MSI in large pan-cancer cohort remains to be analyzed. Methods: We retrospectively reviewed all 18189 solid tumor patients from August 1, 2020 to February 5, 2023 from HaploX Genomic Sequencing Center and analyzed their germline mutations in MMR genes (MLH1,MSH2,PMS2,MSH6 and EPCAM). MSI status was determined using Sanger sequencing or Next-generation sequencing, with tumor MSI status classified as microsatellite instability-high (MSI-H), or microsatellite-stable (MSS). Results: LS-associated germline mutations were detected in 0.313% (n = 57) of patients, and75.4% (n = 43) of patients were identified as MSI-H, and 24.6% (n = 14) of patients were MSS. The patients were grouped into CRC/EC group (colorectal cancer and endometrial cancer) and non-CRC/EC (lung cancer, central nervous system tumor, gastric cancer, esophageal cancer, hepatological cancer, pancreatic adenocarcinoma, small bowel carcinoma, kidney cancer, etc.). Among MSI-H patients, the CRC/EC group accounted for 90.7% (n = 39) and the non-CRC/EC group accounted for 9.3% (n = 4). In MSS patients, the CRC/EC patients accounted for 35.7% (n = 5), and the non-CRC/EC patients accounted for 60% (n = 9). Among patients with MSS, the most frequently mutated gene was MSH6 (n = 9): MSH6 (NM_000179) c.3261dup (p.F1088Lfs*5) mutation was detected in 4 patients, of which 2 patients were paternal and both were diagnosed as colorectal cancer; MSH6 c.718C > T (p.R240*) mutation was detected in 2 patients with available MMR IHC results, and one of them was MSH6 negative in IHC staining. Conclusions: Traditionally, a family history of colorectal cancer or endometrial cancers was the primary clinical feature to diagnose Lynch syndrome. Once it was recognized that Lynch-associated CRCs were microsatellite unstable, tumor testing became an additional tool for identification of Lynch syndrome. Some previous studies also reported Lynch syndrome mutations, primarily MSH6 and PMS2, were present in a few MSS patients. Thus, MSI status or family history as diagnostic factor alone may result in Lynch syndrome under-recognized, and additional NGS test on MMR genes should facilitate more accurate diagnosis.