5-Glyceryl-methylcytosine (5gmC) is an epigenetic modification recently discovered in the genome of Chlamydomonas reinhardtii. It is formed by attaching a glyceryl group of vitamin C to the methyl moiety of 5mC, a process catalyzed by the CMD1 protein. 5gmC has been shown to play a role in promoting active DNA demethylation and photoacclimation. However, the precise localization of 5gmC and its role in gene transcription remain largely unexplored. To efficiently and economically map the distribution of 5gmC across the genome, we investigated the feasibility of nanopore sequencing for identifying this DNA modification. By introducing 5gmC into a set of model strands, significant current decreases associated with 5gmC were consistently observed during nanopore sequencing. This characteristic current signal enables direct identification of multiple 5gmC sites as well as 5gmC across various sequence contexts with reliable accuracy and single-molecule sensitivity. Moreover, we were able to discriminate between 5gmC and 5mC, as 5mC unambiguously increases the ionic current. These results demonstrate the feasibility of nanopore sequencing for mapping 5gmC at the genomic level and provide new insights into the exploration of the roles of 5gmC as a stable epigenetic mark.