Diatoms are eukaryotic microalgae broadly present in freshwater and marine ecosystems. The great ecological interest about diatoms has recently led to increased efforts towards the exploration of the molecular properties of these organisms and the development of molecular tools to study the function of genes. We are using as model system two pennate diatoms, Pseudo-nitzschia multistriata and Pseudo-nitzschia arenysensis. In order to enable molecular investigations in these two species, we have made use of sequence information from transcriptomic data to identify genes that can be used as reference genes in quantitative PCR (qPCR). We have analyzed the expression of a set of genes commonly used as reference genes: histone H4, TBP (TATA binding protein), RPS (30. S ribosomal protein), GAPDH (glyceraldehyde 3-phosphate dehydrogenase), TUB A and TUB B (tubulin α and tubulin β), ACT (actin), CDK A (cyclin dependent kinase A) and COPA (coatomer protein complex, subunit α). The suitability of these genes as references in qPCR has been tested in different conditions and among different strains. We have found that only TUB A, TUB B and CDK A are stable in all the conditions analyzed. These three genes, in addition to ACT and COPA, are good reference genes for P. multistriata, while GAPDH appears to be differentially expressed in the tested conditions in this species. In P. arenysensis, instead, TUB A, TUB B, CDK A, GAPDH, H4 and RPS show the highest levels of stability and can be considered reliable reference genes. © 2013 Elsevier B.V.