β2-microglobulin (B2M), a component of major histocompatibility complex class I, plays an important role in host immune reaction to tumor, and inactivation of B2M is known to contribute to resistance to immunecheckpointblockade(ICB) treatment. To further characterize the B2M alterations in tumors, we analyzed B2Mhotspotmutations in 2765 benign and malignant tumor tissues by Sanger sequencing and found B2Mmutations in 9 (7.5%) microsatellite-unstable (MSU) colorectal cancers (CRCs) and 3 leukemias (0.6-1.3%), but not in other tumors. Targeted sequencing panel analysis for MSU CRCs showed that B2M-mutated MSU CRCs harbored more driver mutations including TP53 than B2M-wild-type MSU CRCs. Of note, bi-allelic B2M alterations, which had been known to be accumulated during ICB treatment, were frequently found (3/9) in ICB treatment-naive CRCs. Clinicopathologic parameters including CD8 + T cell numbers, cancer stages and patients' survival, however, were not significantly different between B2M-mutated and B2M-wild-type MSU CRCs. Our results indicate that B2M mutation abundance is tissue type-specific (e.g., MSU CRCs) and that genetic makeup of B2M mutation might possibly shape the MSU CRC genomes even before the ICB therapies. Our results show that B2M mutation is common in MSU CRCs, which is one of the main targets for ICB treatment, suggesting that frequent B2M mutation status should be reminded for MSU CRCs in patient selection of ICB.