The conventional treatment strategies for patients with metastatic colorectal cancer (mCRC) are predominantly guided by the status of RAS and BRAF mutations. Although patients may exhibit analogous pathological characteristics and undergo similar treatment regimens, notable disparities in their prognostic outcomes can be observed. Therefore, tissue and plasma samples from 40 mCRC patients underwent next-generation sequencing targeting 425 cancer-relevant genes. Genomic variations and canonical oncogenic pathways were investigated for their prognostic effects in association with progression-free survival (PFS) of these patients. We found that patients with BRCA2 and KMT2A mutations exhibited worse prognostic outcomes after chemotherapy-based treatment (univariate, P < 0.01). Further pathway analysis indicated that alterations in the homologous recombination pathway and in the KMT2A signaling network were also significantly associated with shortened PFS (univariate, P < 0.01). Additionally, mutation signature analysis showed that patients with higher proportions of defective mismatch repair (dMMR)-related mutational signatures. Had a worse prognosis (univariate, P = 0.02). KMT2A mutations (hazard ratio [HR], 4.47; 95% confidence interval [CI], 1-19.93; P =0.050) and dMMR signature proportions (HR, 3.57; 95% CI, 1.42-8.96; P = 0.007) remained independently associated with PFS after multivariate analysis and the results were further externally validated. These findings may enhance our understanding of this disease and may potentially facilitate the optimization of its treatment approaches.