Abstract: This study aimed to investigate the evolution of phospholipid composition and the positional distribution of fatty acids during the oxidation of fish oil. Phospholipid composition and the positional distribution of fatty acids on the glycerol backbone in silver carp (Hypophthalmichthys molitrix) fish oil extracted from viscera during incubation at 60 ^oC were characterized by phospholipidomics. The results showed that a total of 373 glycerophospholipids (GP), 39 sphingophospholipids (SP), and 1 sterol lipid were identified in the accelerated oxidation fish oil. The GP components exhibited distinct time-dependent changes: Lysophosphatidic acid (LPA), lysophosphatidylethanolamine (LPC), monomeric phosphatidylcholine (MLCL), phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol (PI), and phosphatidylmethanol (PMe) showed significant increases in content with oxidation time (P<0.05), while bis (2-methylphosphorodicarbonate) (BisMePA) decreased markedly. Phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylethanol (PEt), phosphatidylserine (PS), and cardiolipin (CL) remained stable. Analysis of phospholipid fatty acid composition revealed significant positional specificity in saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA): SFA accounted for 41.79% to 56.91%, MUFA for 24.16% to 38.97%, and PUFA for 8.40% to 33.44%. Notably, Sn-2-positioned PUFA (predominantly C18:2, C18:3, and DHA) showed the highest content, significantly exceeding Sn-1-positioned counterparts (P<0.05). During the oxidation process, MUFA at the Sn-1 site, such as C8:1, C21:1, and C22:1, showed a decreasing trend (P<0.05), while the content of C18:1 and C25:1 showed an increasing trend. Most PUFA at Sn-2 showed a significant downward trend, including C10:2, C11:3, and C18:3. Only the content of C18:2 showed a significant increase (P<0.05). The C18:1 in MUFA exhibited a gradient increasing distribution at Sn-1, Sn-2, and Sn-3/4 positions, and its content at each position significantly increased after oxidation (P<0.05). Notably, SFA distribution remained stable across glycerol backbone positions without significant changes caused by oxidation (P>0.05). In summary, accelerated oxidation in an oven revealed that MUFA at the Sn-1 position and PUFA at the Sn-2 position in silver carp fish oil phospholipids were highly susceptible to oxidation, and their specific degradation mechanisms were closely associated with the positional distribution characteristics of fatty acids. This study offers novel insights into the molecular mechanism of lipid oxidation in aquatic products.