Thiamin (vitamin B1) is mainly produced by bacteria and phytoplankton and then transferred to zooplankton and higher trophic levels but knowledge on the dynamics of these processes in aquatic ecosystems is lacking. Hence, the seasonal variation in thiamin content was assessed in field samples of copepods and in pico-, nano- and micro-plankton of two size classes (0.7–3 µm and > 3 µm) collected monthly in the Baltic Sea during 3 years and in the Skagerrak during 1 year. Copepods exhibited species-specific concentrations of thiamin and Acartia sp. had the highest carbon-specific thiamin content, at both locations. Even members of the same genus, but from different systems contained different levels of thiamin, with higher thiamin content per specimen in copepods from the Skagerrak compared to congeners from the Baltic Sea. Furthermore, our results show that the small plankton (0.7–3 µm) had a higher carbon-specific thiamin content compared to the large (> 3 µm). Additionally, there was a large seasonal variation and thiamin content was highly correlated comparing the two size fractions. Finally, there was an overall positive correlation between thiamin content in copepods and plankton. However, for periods of high thiamin content in the two size fractions, this correlation was negative. This suggests a decoupling between thiamin availability in pico-, nano- and micro-plankton and zooplankton in the Baltic Sea. Knowledge about concentrations of this essential micronutrient in the aquatic food web is limited and this study constitutes a foundation for further understanding the dynamics of thiamin in aquatic environments.