Purpose: The efficacy of immunotherapy varies widely among different gastrointestinal cancers. Response to immune checkpoint inhibitors is shown to correlate with tumor mutation load (TML), mismatch repair deficiency status (dMMR), and programmed cell death-ligand 1 (PD-L1) expression. Herein, we attempt to quantify TML, dMMR, and PD-L1 expression and determine their interrelationship in gastrointestinal cancers. Experimental Design: A total of 4125 tumors from 14 different gastrointestinal cancer sites were studied. Next-generation sequencing was performed on genomic DNA isolated from formalin-fixed paraffin-embedded (FFPE) tumor samples using the NextSeq platform. TML was calculated using only somatic nonsynonymous missense mutations sequenced with a 592-gene panel. Microsatellite instability (MSI) was assessed by validated direct analysis of altered known MSI loci in the target regions of the sequenced genes. PD-L1 expression was analyzed by immunohistochemistry. Results: Right-sided colon and small bowel adenocarcinomas had the highest prevalence of TML-high tumors (14.6% and 10.2%, respectively). Pancreatic neuroendocrine tumors and gastrointestinal stromal tumors (GIST) had the lowest rates of TML-high (1.3% and 0%, respectively). TML-high status was strongly associated with MSI-H (P<0.0001). However, all TML-high anal cancers (8.3%) were microsatellite stable (MSS). Higher PD-L1 expression was more likely to be seen in MSI compared with MSS tumors (20.6% vs. 7.8%, P<0.0001). Conclusions: TML-high rate varied widely among gastrointestinal cancers. Although microsatellite instability is conceivably the main driver for TML-high status, other factors may be involved. Future clinical trials are needed to evaluate whether the integration of TML, MSI, and PD-L1 could better identify potential responders to immunotherapy.