The domestic rainbow trout producers issued a standard with an aquatic association that classified rainbow trout as salmon, which raised the concern of consumers on the fish parasites infection. Herein, an in situ method was developed using "iKnife" and rapid evaporative ionization mass spectrometry based lipidomics for real-time discrimination of salmon and rainbow trout without sample preparation. A total of 12 fatty acids and 37 phospholipid species was identified and imported into statistical analysis for building an in situ and real-time recognition model. The ions with | p(corr)| > 0.5 and | p| > 0.03 were shown to be responsible for allocating samples, and the ions with high correlation values, such as of m/ z 747.50, 771.49, and 863.55, indicated large weights in identification of the salmon and rainbow trout. The results indicated that this technology could be employed as a front-line test method to ensure the authenticity of salmon products.