DNA methylation is one of the most extensively studied epigenetic regulatory mechanisms, known to play crucial roles in various organisms. It has been implicated in the regulation of gene expression and chromatin changes, ranging from global alterations during cell state transitions to locus-specific modifications. 5-hydroxymethylcytosine (5hmC) is produced by a major oxidation, from 5-methylcytosine (5mC), catalyzed by the ten eleven translocation (TET) enzymes, and is gradually being recognized for its significant role in genome regulation. With the development of state-of-the-art experimental techniques, it has become possible to detect and distinguish 5mC and 5hmC at base resolution. Various techniques have evolved, ranging from methods based on bisulfite conversion to bisulfite-free approaches and third-generation sequencing techniques. The role of 5hmC has been primarily studied in the context of global epigenetic reprogramming during developmental processes, as well as in embryonic stem cells (ESCs) and neuronal cells. This review aims to comprehensively report the recent techniques and discuss the emerging roles of 5hmC.