The diatom Didymosphenia geminata can produce thick benthic mats along river bottoms, possibly in association with soluble reactive phosphorus mat-forming genetic variants, or gene expression associated with environmental variation. We isolated cells collected from mat-forming and non-mat-forming sites in British Columbia and Alberta, Canada and compared soluble reactive phosphorus, frequency of dividing cells and gene expression via RNA sequencing. Frequency of dividing cells and soluble reactive phosphorus were higher at the non-mat-forming site and there was no significant difference in frequency of dividing cells between Alberta and British Columbia sites, with one exception. Sixty-six gene products from reference diatom (Phaeodactylum tricornutum and Thalassiosira pseudonana) and bacterial (Caulobacter crescentus) genomes were expressed only in the mat-forming sample and many were related to phosphorus metabolism. One hundred seventy two genes were expressed only in the non-mat-forming sample and many were related to cell division and silica metabolism. Differential gene expression in the periphyton communities suggests that there might be different periphyton molecular phenotypes, including D. geminata, related to P acquisition and polysaccharide metabolism at the low P site and cell division and growth at the high P site.