Background: Microsatellite instability (MSI) is a molecular marker of a deficient in mismatch repair (MMR) system. It is one of the main hallmarks in colorectal cancer (CRC) and gastric cancer (GC), occurring in 15% and 18% of CRC and GC respectively. Mutations occur in MMR system genes will impair DNA mismatch recognition and cause accumulation of mutations in DNA microsatellite regions. As a result, microsatellite instability-high (MSI-H) patients often possess an abnormally high number of somatic mutations and would lead to high tumour mutation burden (TMB), which is a prevalent biomarker in cancer immunotherapy. Here we analysed the genomic landscape of 974 gastrointestinal cancer patients to provide new insights on the relationship between MSI, TMB and other genetic mutation characteristics. Methods: Panel sequencing targeting 680 cancer-related genes were performed on patients with colonrectal cancer (n = 569), gastric cancer (n = 375), intestinal cancer (n = 25) and oesophagal cancer (n = 5) on either fresh tumour tissue or formalin-fixed paraffin embedded tumor DNA. Genomic alterations including single nucleotide variations, short insertions and deletions, copy number variations, tumour mutation burden and micro-satellite instability were analysed. Results: Among the 974 patients, 86 patients (8.83%) were classified as MSI-H based on evaluation of 117 MMR-related loci, while rest of the patients (n = 888, 91.2%) were classified as microsatelite stable (MSS). In MSI-H cohort, 68.6% of the patients were classified as TMB-H with a TMB level higher than 10 Muts/Mb, while in MSS cohort, only 3.71% of the patients were classified as TMB-H. The TMB value of MSI-H cohort is also significantly higher than the MSS cohort (p < 0.0001, two-tailed T-test). Meanwhile, we discovered that proportion of patients with mutated lysine methyltransferase family gene KMT2D (54.65% vs 7.17%, p < 0.0001), KMT2B (48.84% vs 3.25%, p < 0.0001) and KMT2C (44.19% vs 7.17%, p < 0.0001) showed significant difference between MSI-H and MSS cohorts (Two-sided Fisher’s exact test). Conclusions: This study demonstrated that MSI-H is highly correlated with TMB-H, providing a new insight of evaluating gastrointestinal cancer treatment on MSI-H patients. The study also revealed novel MSI-H genetic mutation landscape for KMT gene family. Concurrent with recent researches, this study further boosts the potential of KMT genes to be used as a biomarker in both diagnosis and immunotherapy treatment.