Background: Microsatellite instability/mismatch repair deficiency (MSI/dMMR) has been approved as a biomarker for immune checkpoint inhibitor therapy (ICI) . However, the deficiency of mismatch repair system also caused wide-spreading insertion/deletion (indel) mutations in the exonic microsatellite sites of critical immunotherapy related genes such as β-2-macroglobulin (B2M) whose product is critical to antigen presentation. Cases of aquired resistance to ICI treatment caused by the inactivation of B2M have been reported. Therefore, we investigated the mutational profile of B2M across the MSI/dMMR high prevalent cancers such as colorectal cancer (CRC), gastric cancer (GC) and endometrial cancer (EC). Methods: FFPE tumor samples from 37 CRC, 46 GC and 25 EC patients with matched normal tissues were collected for next-generation sequencing (NGS)-based 450 genes panel assay. Genomic alterations including single base substitution, short and long insertions/deletions, copy number variations, and gene rearrangement and tumor mutational burden were assessed. Immunohistochemistry (IHC) with antibody against B2M were performed on available samples to estimate the expression and localization of these proteins. Results: Exonic microsatellite sites of B2M gene have been found instable in 51% (19/37) of CRC, 22% (10/46) of GC and 8% (2/25) of EC. MSI caused small indels at B2M coding region leads to reading frame shift and the production of nonfunctional truncated proteins. Biallelic frameshift mutations, causing non-functional proteins, were found in 47% (9/19) of CRC and 30% (3/10) of GC patients carrying Indels in B2M gene. IHC assays showed the impaired expression of B2M proteins due to frameshift mutations. In addition, NGS test confirmed concurrent mutation in B2M for a MSI-H CRC patient with primary resistance to forth-line anti-PD1 treatment. Conclusions: The extensive mutations in the coding region of genes caused by the dMMR render the cancer cell sensitive to ICI. However, the concomitant variants in exonic microsatellite sites of B2M gene may compromise the predictability of MSI/dMMR as an ICI biomarker. Biallelic mutation of B2M may cause primary resistance to ICI while the mono-allelic mutation of B2M may gain acquired resistance. Therefore, B2M status could be considered when using MSI/dMMR as a biomarker for ICI.