Thiamine (vitamin B1) deficiency in salmonids related to a lipid-rich fish diet causes offspring mortality in the yolk-sac fry phase. A low free thiamine (THIAM) concentration in eggs is an indication of this syndrome. Thiamine deficiency of salmon (Salmo salar) feeding in the Baltic Sea, called M74, was connected to the principal prey fish and feeding area using fatty acid (FA) signature analysis. The FAs of feeding salmon from two areas of the Baltic Sea, the Baltic Proper (57°10' 19°30') and the Bothnian Sea (61°30' 20°00') in 2004, reflected the principal prey species in these areas, sprat (Sprattus sprattus) and herring (Clupea harengus), respectively. Arachidonic acid (ARA, 20:4n-6) and 18:1n-7 indicated dietary herring, 18:1n-9 dietary sprat and 14:0 feeding in the Baltic Proper. The muscle FA profile of non-M74 female spawners of the River Simojoki in a year (1998) with a moderate M74 incidence and salmon of a non-M74 year (2004) reflected herring FAs, whereas the FAs in the M74 year and specifically in M74 females displayed characteristics of sprat. In the M74 year, the THIAM concentration had the strongest positive correlation with the proportion of muscle ARA, and the strongest negative correlations with 14:0 and the ratios 18:1n-9/ARA and 14:0/ARA. Thus, ARA along with 14:0 and these ratios were the most sensitive FA indicators of the dietary species and origin of the M74 syndrome. Despite the pre-spawning fasting, tissue FA signatures were consequently able to connect dietary sprat in the Baltic Proper with thiamine deficiency in Baltic salmon.