Background: SMAD4 mutations have been associated with poor prognosis in patients with metastatic colorectal cancer. However, results have been inconsistent between studies. This study aimed to determine the impact of coexisting mutations with SMAD4 on overall survival (OS) in patients with metastatic colorectal cancer. Methods: This is a retrospective, single institute study. Patients with metastatic colorectal cancer treated at City of Hope between 2013 and 2020 and with available Clinical Laboratory Improvement Amendment (CLIA) certified next-generation tumor sequencing (NGS) results were included. Overall survival was measured from the date of diagnosis of metastatic colorectal cancer. Differences in OS between groups were compared using Kaplan–Meier curves, with p-values calculated via log-rank test. Both univariate and multivariate Cox regression models were applied to estimate the hazard ratios and confidence intervals of survival based on mutation status and other clinical factors. Results: 433 patients with metastatic colorectal cancer were identified. SMAD4 mutation was found in 16.2% (70/433) of tumors. A systemic univariate and multivariate survival analysis model including age, gender, sidedness of primary tumor, RAS, BRAF^V600E, APC, TP53 and SMAD4 status showed SMAD4 mutation alone was not associated with worse prognosis (multivariate HR = 1.25, 95% CI 0.90-1.73, p = 0.18). We then investigated SMAD4 mutation in combination with each of the top 4 most frequently associated oncogenes or tumor suppressor genes (TP53-80%, n = 56; APC- 59%, n = 41; RAS-54%, n = 38; BRAF^V600E-11%, n = 8) as a predictive biomarker of survival in patients with metastatic colorectal cancer. Coexisting mutations in SMAD4 and TP53 were significantly associated with worse overall survival (multivariate HR = 2.51, 95% CI 1.46-4.30, p = 0.001). The median overall survival of patients with coexisting SMAD4 and TP53 mutation was 24.2 months compared to 42.2 months for the rest of the population (p = 0.002). Conclusions:SMAD4 mutation status alone is not sufficient for predicting poor prognosis in patients with metastatic colorectal cancer. Coexisting mutations in SMAD4 and TP53 were significantly associated with worse overall survival in patients with metastatic colorectal cancer.