There is a growing demand for marine omega-3 fatty acids (FAs) that is produced in high amounts by some microalgae. Here we determined the FA profiles of two cold water adapted diatoms, Chaetoceros wighamii and Thalassiosira baltica. The cultures were acclimated to different temperatures (3, 7, 11, 15, and 19 °C) and irradiance (20, 40, 130, and 450 μmol photons m−2 s−1) and the FA profiles were determined in exponential and stationary growth phases, the latter induced by different nutrient limitation (N, P, and Si). The maximum growth rate was obtained by both species at 11 °C, ≥ 130 μmol photons m−2 s−1 and was 0.8 day−1 and 0.6 day−1 for C. wighamii and T. baltica, respectively. Both species contained relatively high amounts of eicosapentaenoic acid (EPA). Thalassiosira baltica accumulated maximally ~ 30 mg EPA g−1 ash-free dry weight (AFDW) under Si-limitation. The content of docosahexaenoic acid (DHA) was lower, reaching up to 4 mg DHA g−1 AFDW in T. baltica. The concentration of EPA correlated positively with the chlorophyll a:carbon ratio, suggesting that it is bound to membranes in the photosynthetic apparatus and the EPA content in T. baltica was high enough to consider it as a potent candidate for cultivation under cold (< 15 °C) conditions. Covering a wide range of environmental conditions, the strongest differentiation in FA profiles was observed between the species with the growth phase/nutrient limitation pattern as the second most important driver of the FA composition.