5-Hydroxymethylcytosine (C-hm) is an essential intermediate in the active DNA demethylation pathway. Here we report a new base-resolution method for measuring C-hm by combining peroxotungstate-mediated oxidation and sequencing analysis. We reveal that an oxidized product of C-hm, trihydroxylated thymine (T-th.), tolerated the incorporation of dATP as a substrate in the process of DNA polymerase elongation. By comparing the results of Sanger sequencing before and after the oxidation, we observed that hmC sites on single-stranded DNAs could be discriminated from unmethylated cytosines. We found that a thermal cycle condition during peroxotungstate treatment enhanced the oxidation reaction of C-hm in double-stranded DNA. Furthermore, Illumina sequencing analysis of C-hm-containing synthetic genome fragments enabled us to identify simultaneously "the positions of C-hm in base resolution. This bisulfite-free simple hmC detection technique could facilitate the acquisition of epigenomic information.