Background: It is well-established that only patients with wild-type KRAS metastatic colorectal cancer (mCRC) benefit from treatment with an epidermal growth factor receptor monoclonal antibody (anti-EGFR mAb). Recently, in patients with tumor progression after anti-EGFR mAb, occurrence of EGFR mutation (n=2/10) [Montagut C et al., Nat Med 2012] or KRAS mutation (n=6/10) [Misale S et al., Nature 2012] in metastases has been identified. These mutations could explain treatment resistance but still need to be confirmed with simultaneous analysis of KRAS and EGFR mutations. Methods: We analyzed 37 tumor samples after anti-EGFR mAb treatment for mCRC (34 from metastasis lesions and 3 from primary tumors). We analyzed KRAS (codons 12 and 13), BRAF^V600E and EGFR^S492R mutations using a highly sensitive technique, pyrosequencing (TheraScreen KRAS Pyro Kit, Qiagen). All tumors were KRAS, BRAF^V600E and EGFR^S492Rwild-type before anti-EGFR mAb treatment. Results: The majority of patients were treated using anti-EGFR mAb in first-line chemotherapy (70%) and combined with cytotoxic chemotherapy (96%). Cetuximab was used in 86% and panitumumab in 14% of the cases. Among the 37 tumor specimens, 8 were collected after disease progression, and the others after disease control. No EGFR^S492R mutation was detected. No tumors developed BRAF mutation but one tumor acquired a KRAS mutation. Nevertheless, the KRAS mutation in this patient (G12V) was detected, after 5-fluorouracil plus cetuximab therapy, only in the primary tumor in the colon but not in the liver metastasis. Moreover, there was a disease control (partial response). Conclusions: Our results suggest that EGFR^S492R and acquired KRAS mutations during anti-EGFR mAb therapy are not the only factors accounting for anti-EGFR resistance. Moreover, occurrence of KRAS mutation during anti-EGFR therapy could differ between primary tumor and metastases.