Background: Although BRAF V600E accounts for the majority of the BRAF mutations in mCRC, non-V600 BRAF variants have been shown in recent years to represent a distinct molecular subtype of mCRC. This study provides a comprehensive profile of BRAF V600 and non-V600 variants, their clonalities and co-mutations in mCRC using a large genomic database of circulating tumor DNA (ctDNA). Methods: A systematic analysis of Guardant360 results was performed among ctDNA samples of mCRC patients from September 2014 to May 2021. A variant was defined as clonal if the mutant allele frequency (MAF) was greater than 50% of the highest somatic MAF in the sample; otherwise it was defined as subclonal. A previously validated anti-EGFR exposure score was applied to predict prior anti-EGFR therapies. Co-mutation analysis was conducted with BRAF, KRAS, NRAS, NF1, ERBB2, PIK3CA and SMAD4. Results: 1,733 out of 14,742 mCRC patients had at least one BRAF variant, including 6.5% of patients with BRAF V600 variants, 1.1% with class II variants, 1.9% with class III variants, and 3.2% with unclassified variants. 431 unique BRAF variants were identified in a total of 1,905 BRAF variants. 70.7% of BRAF V600 variants were clonal while most (56.0%-78.8%) class II, III and unclassified BRAF variants were subclonal (Table). Patients with non-BRAF V600 variants tend to be younger and male. The prevalence of BRAF class II and III variants were higher (2.1% and 3.7%) in patients with predicted prior anti-EGFR exposure compared with patients predicted to have no prior exposure (0.8% and 1.4%). BRAF variants of all classes are more likely to be subclonal in patients predicted to have anti-EGFR exposure than those predicted nonexposed (p<0.05 in all classes, Fisher’s exact test). Among patients with non-BRAF V600 variants, a greater fraction of co-occurring KRAS and NRAS mutations were detected in those predicted to have prior anti-EGFR exposure. In the patients without predicted EGFR exposure, BRAF class II and III variants showed a higher rate of co-occurring KRAS mutations (25.6% and 21.5%) and co-occurring NRAS mutations (5.8% and 2.7%) compared with BRAF V600 variants (2.4% for KRAS and 0.1% for NRAS); however, co-occurring KRAS G12C was only noted in one patient with a BRAF class II variant. The analysis of outcome data by variant class will be presented at the meeting. Conclusions: We noted significant differences between BRAF V600 and class II/III variants using a large genomic database. Within BRAF class II and III variants, the enrichment in patients with predicted anti-EGFR exposure and the high fraction of co-mutations in KRAS/NRAS suggest a unique therapeutic need for these patients.