Fatty acids (FAs) were analysed in Baltic herring (Clupea harengus membras) stored in the Swedish Environmental Specimen Bank for up to 40 years. The purpose was to evaluate the retrospective use of FA signatures to detect temporal and spatial changes in the Baltic ecosystem. Fish from northern and central Baltic captured in the 1970s, the 1980s, in 1990, 2000, and 2009 and stored at − 25 °C were analysed. From the 1980s and onward herring from the south Baltic were included. A total of 55 FA and 4 alkenyl chains (detected as dimethyl acetals) were identified, and 28 of these (present at > 0.5% by weight) were used in evaluation of the data. The amount of some 20–22 carbon polyunsaturated fatty acids (PUFA) was related to time with lower amounts in older samples while other PUFAs were not related to time. Principal component analysis with saturated FAs and monounsaturated FAs showed similar sample groupings as the one obtained by including the PUFAs. The differences found in herring FA in this longitudinal study could be attributed to location of sampling, year of collection and storage time. However, the clearly distinguishable pattern in the FA composition in herrings from different locations in the Baltic Sea seen at all decades indicate that this technique can be used retrospectively.