The study presents a genome-wide approach, Tet-assisted bisulfite sequencing (TAB-Seq), for mapping 5-hydroxymethylcytosine (5hmC) at base resolution and quantifying its relative abundance. TAB-Seq combines β-glucosylation to protect 5hmC and mTet1-mediated oxidation to convert 5mC to 5caC, allowing for the distinction of 5hmC from unmodified cytosine and 5mC after bisulfite treatment. Applied to human and mouse embryonic stem cells (ESCs), TAB-Seq reveals widespread distribution of 5hmC, sequence bias, and strand asymmetry at 5hmC sites. High levels of 5hmC are observed near transcription factor binding sites, while 5mC levels are low. 5hmC is enriched in distal regulatory elements, with higher levels in CpG-poor regions, and shows a negative correlation with 5mC levels. The TAB-Seq method provides a detailed understanding of 5hmC distribution and abundance, highlighting its dynamic regulation and potential functional roles in gene regulation.The study presents a genome-wide approach, Tet-assisted bisulfite sequencing (TAB-Seq), for mapping 5-hydroxymethylcytosine (5hmC) at base resolution and quantifying its relative abundance. TAB-Seq combines β-glucosylation to protect 5hmC and mTet1-mediated oxidation to convert 5mC to 5caC, allowing for the distinction of 5hmC from unmodified cytosine and 5mC after bisulfite treatment. Applied to human and mouse embryonic stem cells (ESCs), TAB-Seq reveals widespread distribution of 5hmC, sequence bias, and strand asymmetry at 5hmC sites. High levels of 5hmC are observed near transcription factor binding sites, while 5mC levels are low. 5hmC is enriched in distal regulatory elements, with higher levels in CpG-poor regions, and shows a negative correlation with 5mC levels. The TAB-Seq method provides a detailed understanding of 5hmC distribution and abundance, highlighting its dynamic regulation and potential functional roles in gene regulation.