Abstract Colorectal cancers (CRCs) are divided into three different subclasses based on the genetic and epigenetic profiles: CpG island methylator phenotype (CIMP) 1, CIMP2 and CIMP-negative. CIMP1 has been shown to be associated with BRAF mutations and sessile serrated adenoma. CIMP2 has been shown to be associated with KRAS mutation, but precursor lesions of CIMP2 remain to be determined. Our aim of this study is to identify the origin of CRCs with CIMP by examining the promoter methylation of tumor-related genes and genome-wide methylation profiles in precursor lesions. Based on histological examination after endoscopic mucosal resection or operation, the specimens were divided into two groups: 65 serrated polyps (SPs), 109 colon adenomas (ADs) with or without high grade dysplasia. Endoscopically, subsets of SPs were composed of flat and protruded lesions, and biopsy samples were obtained from both lesions, and were divided into three groups: flat lesions only (SP1, 19 cases), flat lesions adjacent to protruded lesions (SP2, 21 cases), or protruded lesions adjacent to flat lesions (SP3, 25 cases). Mutations of BRAF were observed frequently in SPs but not in ADs (26% vs 2%), indicating that the SPs are the origin of CIMP1 characterized by BRAF mutations and microsatellite instability as suggested previously. Frequencies of KRAS mutations were highest in tubulovillous adenomas (TVs) followed by SPs, and tubular adenomas (TAs) (57% vs 43% vs 20%). High frequency of KRAS mutations in SPs and TVs prompt us to examine genome-wide methylation profiles in these tumors. By hierarchical clustering analysis of the MCAM results, SP3, SP1/SP2, and normal colon were grouped into three distinct clusters, and it is suggested that genome-wide methylation occurs during the course of progression from SP2 to SP3, and was associated with acquirement of CIMP. We further examined methylation profiles of additional SPs, ADs (TVs and TAs) with KRAS mutations. Cluster analysis of KRAS mutant SPs and ADs grouped tumors into three clusters; cluster-1 with genome-wide aberrant methylation, cluster-2 with moderate methylation, and cluster-3 with little methylation. Cluster-1 contained SP3 and majority of TVs, cluster-2 contained SP1, SP2, and TAs, and cluster-3 contained normal colon. The present study suggested that CRCs with CIMP2 are originated from two distinct subgroups; CIMP2-protruded (CIMP2-P) originated from serrated-villous protruded tumors, and CIMP2-depressed (CIMP2-D) originated from tubular-depressed pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 169.