Background: Location of colon cancer (right-sided versus left-sided) was reported as a predictor of benefit from cetuximab in the subanalysis of NCIC CTG CO.17 trial. On the other hand, retrospective analyses have identified KRAS including codon61, codon146, BRAF, NRAS, or PIK3CA mutations as potentially negative predictive factors for anti EGFR treatments. This analysis aimed to reveal the prevalence of these mutations in the EGFR pathway according to location of CRC in a large cohort. Methods: Consecutive patients of 1,001 CRC from January 2012 to October 2013 were analyzed in this study. Multiplex genotyping of EGFR pathway was performed on archival samples using Luminex Assay (MABGEN and GENOSEARCH Mu-PACK, MBL) for KRAS including codons 61 and 146, BRAF, NRAS, and PIK3CA. We examined the correlation among mutation profile, clinical data and location of CRC. Statistical analysis was conducted by chi-square test. Results: The incidence of mutations; KRAS codon12 and 13, codon61 and 146, BRAF, NRAS and PIK3CA were 38%, 4.6%, 5.1%, 3.5% and 9.1%, respectively. So called all RAS mutations accounted for 47% in this cohort. The difference of prevalence according to the location of CRC was confirmed in KRAS codon 12 and 13, BRAF. The respective proportion of mutations were 55.9%, 14% for right sided(R), 30.6%, 5.5% for left sided(L), 37.2%, 1% for rectal(RC) CRC (KRAS: R vs L, RC P<0.01, BRAF: R vs L vs RC P<0.01). Conclusions: According to the location of CRC the incidence of KRAS codon 12 and 13, BRAF mutation were different. Increased BRAF mutation in R ,which was strong negative prognostic factor could affect unfavorable outcome of anti EGFR treatment.