Recently, 5-hydroxymethyl cytosine (5-hmC) has been discovered as a naturally existed component of normal mammalian genomic DNAs, and it is generally accepted as the sixth base in the genome. This review will introduce the recent advances in the researches on 5-hmC of its formation, tissue-specific distribution, the roles in cell differentiation and gene expression regulation, and the connections as a epigenetic marker with diseases, such as various cancers. We also summarized the current development of the methodologies to detect methylated or hydroxymetholated cytosines of cells at the genomic levels.