Background: Mutations occurring at the V600 amino acid of BRAF is the most common BRAF mutations in colorectal cancer (CRC), which lead to RAS-independent active monomers (Class 1) and are the targets of BRAF inhibitors. BRAF non-V600 mutants can be further classified as RAS-independent active dimers (Class 2) and RAS-dependent impaired kinase (Class 3). The clinical and genetic distinction of CRC patients carrying different subtypes of BRAF mutations remain to be revealed. Methods: A multicenter retrospective study investigated the mutational profiles of 2,118 CRC patients whose baseline tumor samples were analyzed between June 2015 and June 2020 using capture-based hybrid NGS targeting 400+ cancer-related genes. Clinical characteristics including age, sex, stage, and tumor location were analyzed. A public cBioPortal cohort of 471 metastatic CRC patients was used for survival analysis. Results: A total of 473 patients were identified to contain BRAF mutations and can be sub-grouped into Class 1 (N = 246), Class 2 (N = 29), Class 3 (N = 53), and others (N = 145). All Class 1 BRAF mutations were V600E, while the Class 2 and Class 3 mutations were predominantly G469 (52%) and D594 (56%), respectively. No difference in patient’s age, sex, and stage was observed among BRAF Class 1-3 subgroups, but the anatomical location of the tumor differed among subgroups, particularly between Class 1 and Class 3 BRAF-mutant patients significantly (p = 0.027). Specifically, 39% of tumors carrying Class 1 mutations occurred at the right colon, while the most frequent location of tumors with Class 3 mutations was rectum (58%). Mutational profiling revealed that KRAS and APC mutations were enriched in Class 2/3 compared to Class 1 BRAF-mutant patients, while RNF43 and SMAD4 mutations were more frequently observed in Class 1 patients. Mutations causing the activation of the Wnt or RTK/RAS signaling pathways were also more common in Class 2/3 subgroups comparing to Class 1 patients. Furthermore, based on the analysis of mutation allele frequency, Class 1 BRAF mutations tended to be drivers while Class 2/3 BRAF mutations were more likely to be passengers. In addition, mutational signature profiling showed that the NER signature was enriched in Class 1 BRAF-mutant patients but the APOBEC signature in Class2/3 classes. The tumor mutational burden of Class 2 BRAF tumors was higher than the other two subgroups (median: 10.4 vs 7.6 and 8.4), and microsatellite instability-high tumors were more common in Class 1 (11% vs 7% and 4%). In the cBioPortal cohort, Class 1 BRAF-mutant patients had the worst overall survival whereas Class 3 patients demonstrated the best prognosis. Conclusions: The clinical and genetic features of CRC patients harboring Class 2 and 3 BRAF mutations were different from those carrying Class 1 BRAF mutations in aspects including tumor location, concurrent mutations, mutational signatures, as well as survival outcomes.