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• Loss of autofluorescence could improve the clinical examination of oral mucosal lesions. • Observer-related variability affects the assessment of autofluorescence. • Low interobserver agreement claims for devices able to independently detect autofluorescence. The assessment of loss of tissue autofluorescence (LAF) has been proposed as an adjunct to comprehensive oral examination to enhance the detection of mucosal lesions harbouring dysplasia or carcinoma. The assessment of LAF is not based on completely objectified parameters therefore intraobserver and interobserver variability cannot be neglected alongside the issue of correct interpretation of LAF. The present study evaluated intraobserver and interobserver variability in the clinical assessment of LAF as performed by oral medicine practitioners (OMPs) or general dental practitioners (GDPs). Couples of clinical pictures, acquired under white incandescent dental operatory light and during the assessment of LAF performed by VELscope were retrieved. Four OMPs and eight GDPs were asked to assess the pictures and to score the LAF. Kappa statistics allowed the assessment of intra- and inter-observer related variability. Pictures of 109 lesions representative of all oral mucosal sites and clinical appearances were selected. OMPs had a better intraobserver agreement than GDPs (substantial versus moderate). The moderate (k = 0.506) interobserver agreement observed among both OMPs and GDPs in a 2-score model (positive versus negative), lowered down to poor values only among GDPs when a 3-score or 4-score model (including uncertain judgements) was applied. A good agreement (k>0.8) was never observed and the present results are similar to previously reported data about conventional oral examination. Irrespective of the diagnostic accuracy, the assessment of AF seems not to be able to improve observer-related variability in the clinical assessment of oral mucosal lesions. [ABSTRACT FROM AUTHOR]