Background: Clinical trials have demonstrated improvements in survival with immune checkpoint inhibitors (ICIs) for advanced colorectal cancer patients with MSI-H/dMMR detected using PCR-based assays (PCR) or immunohistochemistry (IHC), respectively. MSI-H can also be assessed by next-generation sequencing (NGS). Evaluation of real-world outcomes among MSI-H patients by NGS treated with ICIs are warranted, particularly when results are discordant between these tests. Methods: The VA National Precision Oncology Program Database was accessed to select veterans with colorectal cancer and an MSI-H biomarker by NGS. Baseline patient variables, disease characteristics, and duration of ICI treatment were obtained from the VA’s Corporate Data Warehouse. Concordance between NGS and IHC or PCR testing was computed, and the response rate and duration of ICI treatment in patients with discordant test results were recorded from chart review. Results: Among 1,276 colorectal cancer patients, 71 (5.6%) were found to have MSI-H by NGS. Of these, 22 (30.1%) received ICI. Among 49 patients who did not receive ICI, 36 had stage I-III disease, 5 had limited performance status, 5 were actively being treated with chemotherapy and 3 had completely resected stage IV disease. Of the 71 patients, 29 had dMMR IHC testing, 8 had MSI-H PCR testing, 1 had both IHC and PCR testing, and 34 patients had only NGS testing. No PCR tests were discordant with NGS but 8 of 29 IHC tests were discordant. Among these 8 patients with discordant IHC MMR and MSI-H by NGS, 5 received pembrolizumab. There were 3 partial responses, 1 stable disease and 1 progressive disease. Durable responses were seen with 3 of 5 patients remaining on therapy without progression at the time of this analysis at a median follow up of 8.5 months. Conclusions: In a cohort of NGS MSI-H colorectal cancer patients, there was a high rate of discordant IHC results. Clinical benefit is seen in patients treated with ICI with discordant testing results, suggesting that NGS testing identifies patients with false negative dMMR IHC testing in the real-world clinical setting.