Rainbow trout (Oncorhynchus mykiss), an economically important fish well-known for its high nutrient content and excellent meat quality, is widely farmed throughout the Northern Hemisphere. To enhance the quality of rainbow trout, the industry has adopted flowing-water ponds, deep-water ponds, and deep-water cage culture modes. However, land-based culture modes often suffer from limited activity space and low water exchange rates. The land-sea relay culture model addresses these issues by providing a better hydrological environment, thereby improving the quality of farmed rainbow trout. Despite these advancements, market challenges persist, including unclear product origins and consumers' inability to distinguish quality differences among culture modes. Therefore, it is crucial to evaluate the differences in nutritional value and flavor quality of rainbow trout across these culture modes and to identify characteristic quality indicators. In this study, rainbow trout without internal organs (with an average weight of 3000 g) were collected in May 2025 from four culture modes: flowing-water ponds (FWP), deep-water ponds (DWP), deep-water cages (DWC), and land-sea relay system (LSR). The flowing-water ponds were located at a site in Zuanxin Lake, Heilongjiang Province; the deep-water ponds were situated at a site in Kashi River, Xinjiang Uygur Autonomous Region; and the deep-water cages were placed at a site in Liujiaxia Reservoir, Gansu Province. In the land-sea relay system, fish were cultured at a site in Yantai, Shandong Province. Sensory evaluation, texture, water holding capacity, water distribution, nutritional content, and flavor of rainbow trout from the four culture modes were analyzed. The results showed that: (1) There were significant differences in the sensory evaluation scores of rainbow trout meat among the different culture modes (P< 0.05). The DWC culture mode received the highest color score, while the FWP culture mode exhibited significantly superior texture scores compared to the other three modes. A Salmon Fan and a chroma meter were used to evaluate the color of fish meat from the different culture modes. The DWC mode displayed the highest color card value, with its h* value significantly lower than that of the other modes (P< 0.05). The water holding capacity of the fish meat in the FWP and DWC culture modes was significantly higher than that of the other two modes (P< 0.05), while the proportion of immobilized water in the flowing-water ponds culture mode was significantly higher than that in the other three culture modes (P< 0.05). Compared with the other three modes, the fish meat from the FWP culture mode had higher hardness, gumminess, adhesiveness, and chewiness. (2) The moisture and ash contents were highest in the FWP culture mode. The fat content was highest in the DWC culture mode. For crude protein content, the LSR culture mode had the highest value in fish meat. Polyunsaturated fatty acid content was significantly higher, and saturated fatty acid levels were lower, in the FWP culture mode compared to the other modes (P< 0.05). The AI and TI indices in the FWP culture mode were significantly lower than those in the remaining three groups. Regarding protein nutritional evaluation, both the AAS (Amino Acid Score) and CS (Chemical Score) values for rainbow trout muscles from all four culture modes exceeded 1, indicating that they represent ideal protein sources. The first limiting amino acid in rainbow trout meat from all four culture modes was methionine (Met) + phenylalanine (Phe). (3) Hx and HxR levels were lowest in the fish meat of the LSR culture mode, and bitterness was also the lowest. The content of umami free amino acids in the meat of rainbow trout cultured in FWP was higher than that in the other three culture modes. The flesh of DWC cultured fish exhibited higher contents of umami-free amino acids and inosine monophosphate. Accordingly, it demonstrated a superior umami taste compared to fish from the other three culture modes. A total of 26 volatile substances (including monomers and dimers) were identified across the modes, including 10 aldehydes, 4 ketones, 6 alcohols, 2 esters, 1 furan, and 3 unknown substances. No significant differences were observed in the concentrations of pentanal, hexanal, and 1-penten-3-ol among the four culture modes (P> 0.05). The DWP culture mode had the richest volatile flavor substances, with the highest content of aldehydes and alcohols. In the LSR culture mode, the contents of nonanal, octanal, and 1-penten-3-one were significantly higher than those in the other three culture modes (P< 0.05), presenting strong fruity and fatty flavors. Based on orthogonal partial least squares analysis and variable importance in projection scores > 1.2, free glutamic acid, free phenylalanine, C18:3n-6, 1-butanol, free methionine, inosine monophosphate, C18:2n-6t, hypoxanthine, and free histidine were selected as important indicators for identifying rainbow trout from the four culture modes. The results indicated that FWP cultured rainbow trout exhibited superior textural properties; the DWP mode yielded a richer profile of volatile compounds; DWC cultured trout demonstrated more desirable muscle color and a stronger umami taste; and the LSR culture system provided improved protein nutrition with lower bitterness. The findings of this study offer empirical support for differentiating quality traits and establish a theoretical foundation for discriminating the origins of rainbow trout based on their cultivation modes.