Fourier-transform infrared (FTIR) spectroscopy was carried out on single colonies of Pediastrum duplex present in air-dried preparations of mixed phytoplankton samples isolated from a eutrophic freshwater lake. FTIR absorption spectra had 12 distinct bands over the wavenumber range 3300–900 cm−1 which were tentatively assigned to a range of chemical groups, including -OH (residual water, wavenumber 3299 cm−1), -CH2 (lipid, 2924), -C=O (cellulose, 1739), amide (protein, 1650 and 1542), >P=O (nucleic acid, 1077) and -C-O (starch, 1151 and 1077). Measurement of band areas identified residual water, protein and starch as the major detectable constituents. Areas of single bands and combined bands of -CH2, -C-O and >P=O species normalized to protein (to correct for differences in specimen hydration and thickness) showed wide variation between colonies, indicating environmental heterogeneity. Correlation analysis demonstrated close statistical associations between different molecular species. Particularly high levels of correlation between bands 3/4 (CH2), 6/7 (amide) and 8/9 (-CH3) was consistent with their joint origin from the same molecular species. The isolation of bands 11 and 12 in the correlation pattern was confirmed by factor analysis, suggesting that variation in the level of starch is statistically unrelated to other macromolecules being monitored. The use of FTIR spectroscopy to characterize an algal micro-population within mixed phytoplankton has potential for future studies on biodiversity and environmental interactions at the species level.