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The cuticle of scorpions (Chelicerata: Arachnida) fluoresces under long-wave ultraviolet (UV) light due to the presence of beta-carboline and 7-hydroxy-4-methylcoumarin in the hyaline layer of the exocuticle. The adaptive significance of cuticular UV fluorescence in scorpions is debated. Although several other chelicerate orders (e.g. Opiliones and Solifugae) have been reported to fluoresce on exposure to UV light, the prevalence of cuticular UV fluorescence has not been confirmed beyond scorpions. A systematic study of living chelicerates revealed that UV fluorescence of the unsclerotized integument is ubiquitous across Chelicerata, whereas only scorpions and horseshoe crabs (Xiphosura) exhibit cuticular UV fluorescence. Scanning electron microscopy and histological sectioning confirmed the presence of a hyaline layer in taxa exhibiting cuticular fluorescence. The hyaline layer is absent in all other chelicerates except sea scorpions (Eurypterida) in which a taphonomically altered hyaline layer, that may have fluoresced under UV light, was observed in exceptionally preserved cuticle. Cuticular UV fluorescence appears to be associated with the presence of a hyaline layer, as has long been recognized in scorpions, and may be plesiomorphic among chelicerates. The presence of a hyaline layer in horseshoe crabs and sea scorpions suggests that several putative functions for cuticular UV fluorescence in scorpions can be discounted.