Mass spectrometry imaging (MSI) is a valuable tool for diagnostics and systems biology studies, being a highly sensitive, label-free technique capable of providing comprehensive spatial distribution of different classes of biomolecules. The application of MSI to the study of endogenous compounds has received considerable attention because metabolites are the result of the interactions of a biosystem with its environment. MSI can therefore enhance understanding of disease mechanisms and elucidate mechanisms for biological variation. Here we present the in situ comparative metabolomics imaging data for analyses of light- and dark-treated retina. A wide variety of tissue metabolites were imaged at a high spatial resolution. These include nucleotides, central carbon metabolism pathway intermediates, 2-oxocarboxylic acid metabolism, oxidative phosphorylation, glycerophospholipid metabolism, and cysteine and methionine metabolites. The high lateral resolution enabled the differentiation of retinal layers, allowing determination of the spatial distributions of different endogenous compounds. A number of metabolites demonstrated differences between light and dark conditions. These findings add to the understanding of metabolic activity in the retina. This article is protected by copyright. All rights reserved.