Trypanosomatids, including the human infectious parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania possess a unique DNA modification in their genomes. This DNA modification, known as base J, is synthesized in a two-step process in which specific thymines are hydroxylated to form hmU, then subsequently glucosylated to form base J. While the hydroxylases involved in the first step have been characterized, previous attempts to identify the glucosyltransferase has been unsuccessful. This elusive glucosyltransferase has now been identified and is known as the base J-associated glucosyltransferase, or JGT. Here we demonstrate the involvement of JGT in base J synthesis. The deletion of both alleles of JGT from the genome of T. brucei generates a cell line that completely lacks base J. Reintroduction of the JGT in the JGT-/- background stimulates base J synthesis. In an in vitro assay, recombinant JGT utilizes UDP-glucose to transfer glucose to hmU in the context of dsDNA. Further in vitro characterization of JGT demonstrates its ability glycosylate hmU within any sequence with no significant change in Km or kcat, suggesting that JGT possesses no DNA sequence specificity. The identification of this unique enzyme and its characterization as a DNA sequence non-specific enzyme has led to the development of a technique using JGT to map the location of hmU throughout the genome. JGT can specifically convert hmU to base J in a genomic sample and the resulting base J can be enriched using an anti-base J antibody pull-down. This technique could be used to map the genomic location of hmU and may help to shed light on the potential functional of this oxidized DNA base. These findings presented here have ultimately led to the identification of a novel glucosyltransferase, a greater understanding of the synthesis of base J in trypansomatids, and the development of a technique that can be used to increase our understanding of epigenetic DNA modifications.