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5-hydroxymethylcytosine (5hmC) is an important epigenetic modification, which has the function of gene regulatory. Current methods for genomic mapping of 5-hydroxymethylcytosine have been limited by either cost, sequencing depth, high DNA input, or lack of single-base resolution. We present an approach called reduced representation 5-hydroxymethylcytosine profiling (RRHP) to map 5hmC sites at the single-base resolution. RRHP can detect sites with low 5hmC abundance, and if it’s combined with RRBS data, 5-methylcytosine and 5-hydroxymethylcytosine can be detected at the same time. RRHP will play a key role in improving our understanding of the complexity of DNA modifications in embryo development, neural cell differentiation and cancer.