We aim to identify the prevalence and the role of the mutation in predicting resistance to anti-EGFR agents in metastatic colorectal cancer (mCRC) patients. We retrospectively reviewed tumor-based next generation sequencing (NGS) results from mCRC patients screened for enrollment in the GO40872/STARTRK-2 clinical trial between July 2019 and March 2021. Then, in patients harboring microsatellite stable (MSS) and wild-type mutant mCRC, we reviewed outcome to treatment with anti-EGFR monoclonal antibodies. A total of 246 mCRC patients were screened. Most of them, 215/220 (97.7%), were diagnosed with MSS mCRC and 112/215 (52.1%) with MSS, and wild-type mCRC. Among the latter, 2/112 (1.8%) had mutant mCRC and both received anti-EGFR monotherapy as third line treatment. In both patients, mutant tumors proved primary resistant to anti-EGFR agent panitumumab monotherapy. Of interest, one of these patients was treated with anti-EGFR agents three times throughout his course of treatment, achieving some clinical benefit only when associated with other cytotoxic agents (FOLFOX or irinotecan). We verified in a clinical real-world setting that mutation is a resistance mechanism to anti-EGFR agents in mCRC. Thus, we suggest avoiding the administration of these drugs to MSS and wild-type N57K mutant mCRC.