The majority of phytoplankton pigment studies are from marine, estuarine and oceanic waters, and commonly use estimates of the ratio between marker pigments and chlorophyll a (chl a) for calculating the contribution of phytoplankton groups to total chl a. In this study, we examined pigment ratios obtained with CHEMTAX processing of field data from a range of tropical and temperate freshwater bodies with contrasting water transparency, depth of the mixed layer, and trophic state. The pigment ratios obtained from processing data from fresh waters corresponded quite well with existing values from pure cultures, and were compared with the marine marker pigment:chl a ratio calculated with CHEMTAX using identical procedures. In deep, stratifying lakes a large variation of some pigment ratios with depth was observed, as well as seasonal variation relating to changes in water column structure. There was considerable variation of average phytoplankton pigment ratios among the freshwater bodies studied. Pigment ratios were significantly correlated to indicators of nutrient availability, to depth of the euphotic zone, or to a proxy of light availability. The substantial variation in marker pigment:chl a ratio confirms that algorithms which account for natural variation of pigments in phytoplankton groups are required for accurate assessment of phytoplankton groups based on marker pigment concentration alone.