In the class Insecta, retinal and 3-hydroxyretinal are used as chromophores of visual pigments, but the absolute structure of the 3-hydroxyretinal chromophore has yet to be clarified. This study investigates the chirality of 3-hydroxyretinal in the compound eyes of five representative orders of insects. In the orders Odonata, Hemiptera, Neuroptera and Lepidoptera, and suborders Nematocera and Brachycera of the Diptera, only (3R)-3-hydroxyretinal isomers were detected, but dipterans of the suborder Cyclorrhapha (higher flies) had the (3S)-11-cis enantiomer and a mixture of (3R)-all-trans and (3S)-all-trans 3-hydroxyretinal enantiomers; the ratio of the (3R) enantiomer to the sum of both enantiomers of the all-trans isomer was in the range 9-32%. Despite differences in feeding habits, including one species that is a butterfly parasite, all higher flies analysed to date share the same pattern of 3-hydroxyretinal enantiomers, making them a unique group with regard to the nature of the visual pigment chromophore.