Background: TP53 mutation is present in approximately 50% of metastatic colorectal cancer (CRC). The spectrum of the TP53 mutations is extremely broad including approximately 80% missense mutations. Several missense mutations have been found to possess gain-of-function (GOF) properties in cell line and animal studies, however, confirmation of the concept of GOF in human malignancies is still lacking. Methods: We investigated the impact of TP53 GOF mutations in patients with metastatic CRC using the NGS data within Kaiser Permanente Northern California (KPNC), a large integrated healthcare system. Results: From November 2017 to January 2021, genomic profiling by StrataNGS was performed on 8658 patients, with 1056 patients being metastatic CRC, among whom 740 patients harbored a TP53 mutation (TP53mut) and 316 patients had wild-type TP53 (TP53wt). Ras (KRAS and NRAS) and BRAF mutation appropriately discriminated the overall survival (OS) of patient populations with either TP53wt or TP53mut, confirming the validity of our dataset. We identified seven GOF TP53 mutations (R175H, R248W, R248Q, R249S, R273H, R273L, R282W) in these CRC patients. We show that different GOF mutation differentially impacts the OS. Patients whose CRC harbored TP53mut R248W, R249S, and R282W (poor prognostic TP53mut, N = 47) had significantly worse OS versus patients whose CRC harbored TP53mut R248Q, R175H, R273H and R273L (N = 160, median OS 29.4 vs 44.2 months, HR 0.47, p = 0.007). The OS of the poor prognostic TP53mut patients was also significantly inferior compared to patients whose CRC harbored all other TP53 mutations (N = 1099, median OS 50.1 months, HR 0.55, p = 0.01) or TP53wt (N = 316, median OS 47,5 months, HR 0.54, p = 0.01). The demographics and the percent of Ras, BRAF, and PI3KCA mutations were similar except that the patients with the poor prognostic TP53mut had significantly higher percent of Ras mutation compared to the rest of the GOF TP53mut patients (p = 0.035). When compared to R248Q alone, R248W confers worse OS (median OS 36.3 vs 63.2 months, p = 0.05). Conclusions: Our data suggest that different TP53 GOF mutations are associated with very different clinical outcomes. Additional studies identifying specific TP53 GOF mutations that impact outcomes may provide further insight for drug development and clinical trial design.