DNA methylation (mainly at 5-methylcytosine, 5mC) plays an essential role in embryonic development and cellular biology. Alterations in DNA methylation are associated with disease development, especially hematologic malignancies. To investigate the potential of 5mC for diagnosis and treatment, accurate determination of 5mC is essential. Standard bisulfite sequencing-based methodologies or various optical/electrochemical biosensors for identifying 5mC have limitations, such as high cost, severe DNA degradation, over-estimation of the true 5mC level, being able to only display the average 5mC level, etc. Here we propose a single-molecule strategy for the direct identification of whole-genome 5mC by the combination of DNA fiber-based fluorescence in situ hybridization (DNA fiber FISH) and atomic force microscopy (AFM). Using extended DNA fibers and anti-5mC antibody for the detection of 5mC, it is possible to map the physical location of 5mC within the genome DNA. Together with AFM, this method can present the morphology of anti-5mC-DNA complexes and detailed spacing distribution of two neighboring 5mC sites on a single DNA molecule. Furthermore, this approach can be used for reporting other epigenetic modifications, not limited to 5mC or one single epigenetic modification. It can be anticipated to contribute to the development of clinical diagnosis of epigenetic-related diseases.