Recent studies have shown two distinct non-CIMP methylation clusters in colorectal cancer, raising the possibility that DNA methylation, involving non-CIMP genes, may play a role in the conventional adenoma-carcinoma pathway. A total of 135 adenomas (65 left colon and 70 right colon) were profiled for epigenome-wide DNA methylation using the Illumina HumanMethylation450 BeadChip. A principal components analysis was performed to examine the association between variability in DNA methylation and adenoma location. Linear regression and linear mixed effects models were used to identify locus-specific differential DNA methylation in adenomas of right and left colon. A significant association was present between the first principal component and adenoma location (P=0.007), even after adjustment for subject age and gender (P=0.009). A total of 168 CpG sites were differentially methylated between right- and left-colon adenomas and these loci demonstrated enrichment of homeobox genes (P=3.0 × 10(-12)). None of the 168 probes were associated with CIMP genes. Among CpG loci with the largest difference in methylation between right- and left-colon adenomas, probes associated with PRAC (prostate cancer susceptibility candidate) gene showed hypermethylation in right-colon adenomas whereas those associated with CDX2 (caudal type homeobox transcription factor 2) showed hypermethylation in left-colon adenomas. A subgroup of left-colon adenomas enriched for current smokers (OR=6.1, P=0.004) exhibited a methylation profile similar to right-colon adenomas. In summary, our results indicate distinct patterns of DNA methylation, independent of CIMP genes, in adenomas of the right and left colon.Modern Pathology advance online publication, 19 July 2013; doi:10.1038/modpathol.2013.104.