Abstract: This study aimed to compare the differences in muscle quality and flavor between farmed and wild Japanese eels (Anguilla japonica). Japanese eels from recirculating aquaculture systems (RAS) body weight: (352.5 ± 8.41) g, earth pond culture body weight: (356.32 ± 23.35) g, and wild populations body weight: (344.63 ± 12.44) g were sampled for a systematic comparison. Indicators including muscle texture properties, nutritional composition, content of earthy-musty odor compounds, and volatile flavor compounds were analyzed. The results showed that wild eels exhibited the best texture properties in terms of muscle hardness, springiness, and resilience, and their moisture and crude protein contents were significantly higher than those of RAS-farmed eels. Conversely, RAS-farmed eels had the highest crude fat content. Geosmin analysis revealed that the geosmin content in wild eels was significantly lower than that in farmed eels. In terms of amino acids, the contents of leucine, isoleucine, alanine, and glycine in wild eels were significantly higher than those in farmed eels. However, the ratios of EAA/TAA, EAA/NEAA, and the essential amino acid scores (EAAI) of the three groups all met the FAO/WHO standards, indicating they are all high-quality protein sources. Wild eels had a higher content of ∑n-6 PUFA, while farmed eels provided higher contents of ∑n-3 PUFA, total PUFA, EPA, and DHA. Volatile compound analysis showed that wild eels contained p-xylene, which contributes to the roasted fish flavor, whereas earth pond-cultured eels contained 2-pentylfuran, which contributes to beany and earthy off-flavors. The study demonstrated that Japanese eels from the three sources showed significant differences in muscle texture and flavor, but similar protein quality. Additionally, farmed eels had advantages in fatty acid composition. This research provides data support for the optimization of Japanese eel farming practices and offers a feasibility basis for replacing wild eels with farmed ones, contributing to the conservation of wild eel resources.