Abstract: Lipid metabolism plays a fundamental role in the growth, development, and health maintenance of cold-water fish. This study aimed to characterize lipid deposition patterns and the molecular regulatory mechanisms in the liver, heart, and spleen of Oncorhynchus mykiss at three size classes(350±20) g, (1.8±0.2) kg, and (3.8±0.1) kg, and to identify key candidate genes involved in lipid deposition. RNA-Seq was performed to analyze the transcriptomes of liver, heart, and spleen tissues from fish of different size classes. Histological characteristics of lipid deposition were further observed using hematoxylin-eosin (HE) staining. Lipid deposition exhibited pronounced tissue specificity. The liver was identified as the primary organ for lipid synthesis and storage, with lipid droplets progressively accumulating as body size increased. Only a small number of lipid droplets were observed in the spleen, whereas no obvious lipid deposition was detected in the heart. Comparative analysis among tissues showed that pparg, fabp3, and acox1 were highly expressed in the liver, indicating that lipid metabolic regulation was predominantly concentrated in this organ. The spleen and heart exhibited a developmental transition from processes related to structural maintenance at the early stage to the activation of innate immune pathways at the later stage. In the liver, the PPAR signaling pathway showed pronounced dynamic changes, accompanied by active regulation of genes involved in fatty acid uptake, transport, activation, and oxidation. At the intermediate size class (1.8±0.2) kg, lipid transport was relatively limited, whereas lipid oxidation was enhanced, as indicated by the upregulation of pparb and cpt1. In the large-size class (3.8±0.1) kg, increased expression of pparg, fatp, and fabp suggested a metabolic shift from energy supply toward lipid accumulation and structural lipid biosynthesis with increasing body size. This study reveals that lipid allocation in triploid rainbow trout exhibits significant tissue specificity and stage-dependent characteristics, with key genes including pparg, fabp3, and acox1 in the PPAR signaling pathway constitute core regulatory components of lipid metabolism. These findings provide theoretical insights into lipid nutritional regulation and healthy aquaculture of fish.