The pollution not only in marine but also in freshwater ecosystems rises due to the increasing amount of chemicals in use today. Many substances, such as bisphenol A, are lipid soluble and can thereby affect the lipid content and distribution in tissue. Because of severe consequences for animal and human health, there is a need to develop a method to investigate the influence of environmental contaminants on the lipid distribution of different organs. Here we show a MALDI mass spectrometry (MS) imaging method including a sample preparation protocol encompassing embedding medium and cryosectioning for two model organisms, zebrafish and daphnia. Due to using a high resolution MALDI imaging method the distribution of lipids in different brain regions as well as in non-neuronal tissues of the zebrafish could be acquired in both ionization polarities. Furthermore sample preparation for MS imaging of daphnia cryosections was developed for the first time. Several matrix compounds and solvents were compared to optimize ionization efficiency which resulted in improved signal intensities. In conclusion, the observed phospholipid signatures are an ideal basis for the analysis of changes in lipid pattern caused by (environmental) stress factors. This is the first step to investigate molecular effects of environmental pollutants in aquatic organisms.