CHEMTAX is a matrix factorization program used to derive taxonomic structure of phytoplankton from photosynthetic pigment ratios. The program was originally developed from and applied to the analysis of oceanic phytoplankton assemblages. We found that application of the original CHEMTAX reference matrix to southeastern United States estuarine systems produced inaccurate results, as verified by microscopy. Modification of the matrix, based primarily on the pigment ratios of 33 estuarine isolates, unproved the predictive capabilities of CHEMTAX for our samples. Limitations of the method included an overestimation of diatom biomass (due to the inability to differentiate diatoms from taxa with chloroplasts derived from diatom endosymbionts, notably some dinoflagellates) and a tendency to exclude some raphidophyte species. In complement with microscopic verification, the method was shown to improve assessment of phytoplankton taxonomic composition. © 2005 Estuarine Research Federation.