A new technique enables genome-wide detection and quantification of the epigenetic mark 5-hydroxymethylcytosine (5hmC) in single cells. Based on a method for bulk 5hmC sequencing, 5hmC marks are glucosylated using the T4 phage β-glucosyltransferase, then cut by the restriction enzyme AbaSI. Digested genomic DNA is ligated to double-stranded adapters containing a 2-nucleotide random 3′ overhang, together with a cell-specific barcode, an Illumina 5′ adapter and a T7 promoter. In vitro transcription is used to amplify the DNA fragments linearly in a strand-specific orientation, and the amplified RNA is fragmented and undergoes directional RNA library preparation. When applied to mouse embryonic stem cells, a median of 44,000 unique 5hmC sites per cell was detected. Substantial cell-to-cell variability existed between the number of 5hmC sites on the two strands of the same chromosome. Differences in age between the strands of a chromosome could explain the 5hmC strand bias, as confirmed by a new stochastic model.