The nutritional condition of juvenile mussels appears to play a major role in their performance after they are seeded into coastal mussel farms. However, the extent to which the nutritional condition of juvenile mussels varies with size and starvation are poorly understood. Therefore, this study measured the biochemical composition of juvenile green-lipped mussels of five sizes (range of 0.5–7.5 mm shell length), from three different sources (i.e., harvested from wild, hatchery, coastal floating upweller nursery system) and when subjected to starvation for up to two weeks. The biochemical composition of spat sampled on their arrival from all sources was dominated by protein (ranging from 24 to 70% dry tissue mass), followed by lipid (0.3–9.3%) and carbohydrate (0.3–2.8%). Subsequent starvation treatments predominantly affected carbohydrate content. Initially, spat of 1.1–1.4 mm (harvested wild spat) had the lowest initial carbohydrate content 73% lower than spat of 0.5–1.0 mm (hatchery spat) and 53–65% lower than spat of 1.5–2.0, 2.1–3.4 and 3.5–7.5 mm (hatchery spat that were reared to larger sizes in a floating upweller system). Following one and two weeks of starvation, carbohydrate energy reserves of spat from all size classes showed a decrease of 15–42% from their initial level, except spat of 1.1–1.4 mm (wild) which did not decrease with starvation. Fatty acid dietary markers sampled from both spat of 0.5–1.0 mm (hatchery) and spat of 1.1–1.4 mm (wild) prior to starvation indicated that diatoms were important contributors to the diet of spat from both sources. Following two weeks of starvation, spat from both groups utilised SFA 14:0 and PUFAs, including essential fatty acids EPA, as energy sources, but they conserved AA and DHA. The findings of this study suggest that carbohydrate is the main energy reserve utilised by green-lipped mussel spat when food supplies are limited. Therefore, improving the carbohydrate reserves of spat through appropriate nursery feeding regimes has the potential to improve their resilience to poor feeding conditions following seeding onto coastal mussel farms. •Proximate composition of juvenile mussels 0.5–7.5 mm were dominated by protein.•Juvenile mussels primarily utilize carbohydrate reserve when starved for two weeks.•Juvenile mussels sourced from wild were in poor nutritional condition.•Fatty acids from diatoms are important in both wild and hatchery juvenile mussels.•Nursery culture of juvenile mussels should target improving carbohydrate reserves.