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Blue mussels (Mytilus edulis L. 1758) are important components of coastal ecosystems and in the economy of rural and coastal areas. The understanding of their physiological processes at key life stages is important both within food production systems and in the management of wild populations. Lipids are crucial molecules for bivalve growth, but their diversity and roles have not been fully characterised. In this study, traditional lipid profiling techniques, such as fatty acid (FA) and lipid class analysis, are combined to untargeted lipidomics to elucidate the lipid metabolism in newly settled spat fed on a range of diets. The evaluated diets included single strains treatments (Cylindrotheca fusiformis CCAP 1017/2 –CYL, Isochrysis galbana CCAP 927/1– ISO, Monodopsis subterranean CCAP 848/1 –MONO, Nannochloropsis oceanica CCAP 849/10– NANNO) and a commercial algae paste (SP). Spat growth was influenced by the diets, which, according to their efficacy were ranked as follows: ISO>NANNO/CYL>SP>MONO. A higher triacylglycerols (TG) content, ranging from 4.23±0.82 μg mgashfree Dry weight (DW)-1 at the beginning of the trial (T0) to 51±15.3 μg mgashfreeDW-1 in ISO, characterised significant growth in the spat, whereas, a reduction of TG (0.3±0.08 μg mgashfreeDW-1 in MONO), mono unsaturated FA–MUFA (from 8.52±1.02 μg mgFAashfreeDW-1 at T0 to 2.81±1.02 μg mgFAashfreeDW-1 in MONO) and polyunsaturated FA–PUFA (from 17.57±2.24 μg mgFAashfreeDW-1 at T0 to 6.19±2.49 μg mgFAashfreeDW-1 in MONO) content characterised poor performing groups. Untargeted lipidomics evidenced how the availability of dietary essential PUFA did not influence only neutral lipids but also the membrane lipids, with changes in lipid molecular species in relation to the essential PUFA provided via the diet. Such changes have the potential to affect spat production cycle and their ability to respond to the surrounding environment. This study evidenced the advantages of coupling different lipid analysis techniques, as each technique disclosed relevant information on nutritional requirements of M. edulis juveniles, expanding the existing knowledge on the physiology of this important species. [ABSTRACT FROM AUTHOR]