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Tracement of the origin of seafoods and aquatic products is an important issue for avoiding the falsification of production regions of aquatic products. Herein, by analyzing black tiger shrimp (Penaeus monodon) as a model of aquatic products, we demonstrated a method to trace the origin of aquatic products by analyzing the gut microbiome of black tiger shrimp (Penaeus monodon) collected from three commercial farms in China, via matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Multivariate analysis was executed on the obtained mass spectrometric datasets of bacteria extracted and collected from shrimp samples with different production regions. The gut microorganisms of black tiger shrimps can be identified by scoring the gut microbiota of black tiger shrimps collected from three producing areas. The black tiger shrimp from three production regions were found with distinguished characteristics of bacterial prevalence in their digestive gland microbiota. The dominant bacteria genus in digestive gland microbiota from black tiger shrimp samples from the three production regions are completely different. Single-blind shrimp samples with unknown production region was successfully screened by comparing the detected MS data in different ways. This strategy can trace the production region of the analyzed shrimp samples in a quick and efficient mode. The application of the present protocol was further demonstrated by analyzing the biometric species from gill tissue of Chinese mitten crab (Eriocheir sinensis) samples collected from crab-producing areas in Jiangsu Province (China), that is, Taihu Lake, Gaoyou Lake, and Hongze Lake for tracing their production area. It is expected to extend the presently developed method to provide clues for the origin traceability of other aquatic products. • Tracement of the origin of seafoods is an important issue to avoid the falsification. • A method to trace the origin of aquatic products via MALDI-TOF MS was proposed. • The dominant bacteria in digestive gland microbiota from shrimps are different. • The origin of single-blind shrimp samples can be successfully traced. • The present method holds promise for the origin traceability of aquatic products. [ABSTRACT FROM AUTHOR]