Abstract. The need to acquire a representative periphytic diatom sample for river water quality monitoring has been recognised in the development of existing diatom indices, important in the development and employment of diatom monitoring tools for the Water Framework Directive. In this study, a nested design with replication is employed to investigate the magnitude of variation in diatom biomass, composition and Trophic Diatom Index at varying scales within a small chalk river. The study shows that the use of artificial substrates may not result in diatom communities that are typical of the surrounding natural substrates. Periphytic diatom biomass and composition varies between artificial and natural substrates, riffles and glides and between two stretches of the river channel. The study also highlights the existence of high variation in diatom frustule frequency and biovolume at the individual replicate scale which may have implications for the use of diatoms in routine monitoring.