Much effort in cancer research has focused on the tiny part of our genome that codes for mRNA. However, it has recently been recognized that microRNAs also contribute decisively to tumorigenesis. Studies have also shown that epigenetic silencing by CpG island hypermethylation of microRNAs with tumor suppressor activities is a common feature of human cancer. The importance of other classes of non-coding RNAs, such as long intergenic ncRNAs (lincRNAs) and transcribed ultraconserved regions (T-UCRs) as altered elements in neoplasia, is also gaining recognition. Thus, we wondered whether there were other ncRNAs undergoing CpG island hypermethylation-associated inactivation in cancer cells. We focused on the small nucleolar RNAs (snoRNAs), a subset of ncRNA with a wide variety of cellular functions, such as chemical modification of RNA, pre-RNA processing and control of alternative splicing. By data mining snoRNA databases and the scientific literature, we selected 49 snoRNAs that had a CpG island within ≤ 2 Kb or that were processed from a host gene with a 5'-CpG island. Bisulfite genomic sequencing of multiple clones in normal colon mucosa and the colorectal cancer cell line HCT-116 showed that 46 snoRNAs were equally methylated in the two samples: completely unmethylated (n = 26) or fully methylated (n = 20). Most interestingly, the host gene-associated 5'-CpG islands of the snoRNAs SNORD123, U70C and ACA59B were hypermethylated in the cancer cells but not in the corresponding normal tissue. CpG island hypermethylation was associated with the transcriptional silencing of the respective snoRNAs. Results of a DNA methylation microarray platform in a comprehensive collection of normal tissues, cancer cell lines and primary malignancies demonstrated that the observed hypermethylation of snoRNAs was a common feature of various tumor types, particularly in leukemias. Overall, our findings indicate the existence of a new subclass of ncRNAs, snoRNAs, that are targeted by epigenetic inactivation in human cancer.