AbstractAimsMaternally inherited diabetes and deafness (MIDD) is a rare form of adult‐onset diabetes that can be difficult to diagnose due to its variable clinical phenotype. Transfer RNA‐derived small fragments are a novel, emerging class of small non‐coding RNAs (sncRNAs) that have significant potential as serum biomarkers due to their stress‐induced generation, abundance, stability and ease of detection.MethodsWe investigated the levels of tiRNA 5'ValCAC (alone and in combination with miR‐23b‐3p) identified from small RNA sequencing studies in serum samples from healthy controls, type 1 diabetes, type 2 diabetes and MIDD subjects.ResultsSerum levels of 5'ValCAC were reduced in MIDD and type 2 diabetes subjects compared to controls. Type 2 diabetes subjects had higher serum levels of miR‐23b‐3p compared to all other subjects. Receiver Operating Characteristic analysis showed the potential of 5'ValCAC and miR‐23b‐3p as MIDD biomarkers, with the combination showing excellent separation from type 2 diabetes subjects.ConclusionsThis is the first report showing altered serum levels of tiRNAs in diabetes subjects. The combined use of 5'ValCAC and miR‐23b‐3p as serum biomarkers could potentially differentiate between MIDD subjects and type 2 diabetes subjects.