<jats:p>Iron availability limits primary productivity in large areas of the world's oceans. Ascertaining the iron status of phytoplankton is essential for understanding the factors regulating their growth and ecology. We developed an incubation‐independent, molecular‐based approach to assess the iron nutritional status of specific members of the diatom community, initially focusing on the ecologically important pennate diatom <jats:italic>Pseudo‐nitzschia</jats:italic>. Through a comparative transcriptomic approach, we identified two genes that track the iron status of <jats:italic>Pseudo‐nitzschia</jats:italic> with high fidelity. The first gene, ferritin (<jats:italic><jats:styled-content style="fixed-case">FTN</jats:styled-content></jats:italic>), encodes for the highly specialized iron storage protein induced under iron‐replete conditions. The second gene, <jats:italic><jats:styled-content style="fixed-case">ISIP</jats:styled-content></jats:italic>2a, encodes an iron‐concentrating protein induced under iron‐limiting conditions. In the oceanic diatom <jats:italic>Pseudo‐nitzschia granii</jats:italic> (Hasle) Hasle, transcript abundance of these genes directly relates to changes in iron availability, with increased <jats:italic><jats:styled-content style="fixed-case">FTN</jats:styled-content></jats:italic> transcript abundance under iron‐replete conditions and increased <jats:italic><jats:styled-content style="fixed-case">ISIP</jats:styled-content></jats:italic>2a transcript abundance under iron‐limiting conditions. The resulting <jats:italic><jats:styled-content style="fixed-case">ISIP</jats:styled-content></jats:italic>2a:<jats:italic><jats:styled-content style="fixed-case">FTN</jats:styled-content></jats:italic> transcript ratio reflects the iron status of cells, where a high ratio indicates iron limitation. Field samples collected from iron grow‐out microcosm experiments conducted in low iron waters of the Gulf of Alaska and variable iron waters in the California upwelling zone verify the validity of our proposed <jats:italic>Pseudo‐nitzschia</jats:italic> Iron Limitation Index, which can be used to ascertain in situ iron status and further developed for other ecologically important diatoms.</jats:p>