Discriminative analysis between each zooplankton individual was achieved on the basis of observed total lipid contents and their fatty acid compositions obtained by pyrolysis–gas chromatography combined with on-line methylation using tetramethylammonium hydroxide (TMAH). Pyrolysis in the presence of TMAH allowed the highly sensitive detection of fatty acids in one zooplankton individual as their methyl esters on the resulting pyrogram. The distributions of each saturated and monounsaturated fatty acid, estimated with relative standard deviations of less than 4.5%, were in good agreement with those determined by conventional methylation followed by GC using powdered plankton samples prepared by cryo-milling homogenization. The peak intensities of fatty acid components on the pyrograms were successfully used to discriminate each plankton individual, which differ in the food concentrations, without applying any complicated pre-treatment. The results obtained suggest that plankton individuals cultured in higher food concentrations contain lipid contents between 7.3 and 8.9% m/m whereas those cultured in lower food concentrations lie between 3.2 and 5.9% m/m.