5-hydroxymethylcytosine (5 hmC), the oxidized form of 5-methylcytosine (5 mC), is a base modification with emerging importance in biology and disease. However, like most epigenetic elements, it is transparent to many conventional genetic techniques and is thus challenging to probe. Here, we report a rapid solid-state nanopore assay that is capable of resolving 5 hmC with high specificity and sensitivity and demonstrate its utility in assessing global modification abundance in genomic DNA.