Benthic diatom samples were collected from 45 potential reference river sites in Ireland. Diatom samples were also collected from 24 lowland eutrophic sites. The main objective for sampling the potential reference sites was to determine the type-specific biological typologies represented by these sites. The lowland sites were sampled as part of another project and are used here for comparative purposes. A larger number of taxa was identified from the reference sites (175 species) in comparison with the lowland sites. (71 species). Distinct differences in diatom distribution and diversity between the reference sites and the lowland sites were revealed by DCA analysis. DCA analysis also showed that the original typological classification of the reference sites was not reflected to any extent in the distribution of benthic diatoms between these sites. However, there was some differentiation between the reference river sites with respect to diatom distribution along axis 1 of the DCA ordination and this was strongly correlated with water conductivity.