not-yet-known not-yet-known not-yet-known unknown Background: IgE-mediated fish allergy has long been considered an umbrella term due to the high cross-reactivity of parvalbumin, the major fish allergen. Yet, clinical tolerance to certain fish highlights allergenicity differences. In this study, we sought to construct a fish allergenicity ladder and identify fish parvalbumin epitopes to improve the diagnosis of fish allergy. Methods: Reported clinical history and the serum-specific IgE (sIgE) responses of 200 fish allergic patients were collected and analyzed, while the relative parvalbumin content in different fish were measured for the construction of fish allergenicity ladder. Double-blind placebo-controlled food challenge (DBPCFC) and open challenge against salmon, grass carp and grouper were performed in 58 selected patients for validation of the ladder. Epitope mapping was performed by peptide array against parvalbumins of salmon (both β-1 and β-2), cod, grouper, and grass carp with sera from fish allergic (n=11), partial fish tolerant (n=12), and complete fish tolerant (n=5) patients diagnosed based on oral food challenge outcome. Results: The distribution pattern of clinical, sIgE and molecular data and their strong positive correlation led to the construction of a 4-step fish allergenicity ladder comprising: step 1 of the least allergenic fishes (tuna, halibut, salmon), steps 2 (cod) and 3 (herring and grouper) of moderately allergenic fishes to step 4 of highly allergenic fishes (catfish, grass carp and tilapia). Epitope mapping revealed one epitope from grouper parvalbumin (AA64-78) for diagnosing general fish allergy and one epitopic region from salmon parvalbumin (AA19-33) as biomarker of specific fish tolerance. Only epitope-specific IgE differentiated these patients but not sIgE to fish extract or parvalbumin. Conclusion: The fish ladder and epitopes discovery can precisely differentiate fish-allergic and tolerant subjects and guide fish reintroduction by stepping up the ladder, which innovate fish allergy care in the next millennium.