5-Hydroxymethyluridine is a modified nucleoside where a hydroxymethyl group is attached to the 5-position of the uracil base. It is a naturally occurring modified ribonucleoside found in transfer RNA (tRNA) of various organisms, including bacteria and eukaryotes. Its presence in tRNA is associated with specific structural and functional roles in translation. This compound serves as a crucial biomarker and reference standard in biomedical research. It is a primary oxidative metabolite of thymidine, and its elevated levels in human urine are considered a significant biomarker for assessing oxidative stress and DNA damage. Consequently, it is widely used in studies related to aging, cancer, and other diseases linked to oxidative damage. As a modified nucleoside, 5-hydroxymethyluridine is also of interest in the field of epitranscriptomics, which studies RNA modifications and their biological functions. It is utilized as a chemical standard in analytical methods such as liquid chromatography-mass spectrometry (LC-MS) for the precise quantification of RNA modifications and oxidative DNA/RNA damage products in biological samples.