5-Hydroxymethylcytosine (5hmC) is beginning to be expected to play a role as a diagnostic and prognostic marker of diseases. On the other hand, we developed the methylated-site display-amplified fragment length polymorphism (MSD-AFLP), an affordable, large-scale (approximately 40,000-50,000 CpG sites), highly sensitive methylation profiling method, and applied it to environmental health and disease biomarker discovery research. Herein, we attempted to modify the MSD-AFLP method to detect 5hmC. To validate this method, we compared hydroxymethylation levels among tissues using mouse samples to determine whether this method could detect tissue-specific differential 5hmC. We also considered combining this method with next generation sequencing (NGS). Comparisons using AFLP revealed that in some sites, the variation in hydroxymethylated DNA was greater than that in methylated DNA between tissues. Therefore, we determined the hydroxymethylation levels at these sites using Glucosylation-mediated restriction enzyme sensitive qPCR (gRES-qPCR) to confirm the accuracy of the AFLP analysis. The differences in hydroxymethylation levels between tissues were similar between the two methods. The protocol for combination with NGS was evaluated by comparing the AFLP data with 11 DNA fragments. The differences in hydroxymethylation levels between tissues were similar between the two methods. Cluster analysis demonstrated that NGS data were comparable to AFLP data in detecting differential and contrasting hydroxymethylation patterns across tissues. This method, based on the MSD-AFLP technique, will contribute to various epigenetics-based research, including the discovery of biomarkers and therapeutic drug targets.