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Background: The current diagnostics of fish allergy lack sufficient accuracy such that more reliable tests such as component-resolved diagnosis (CRD) are urgently needed. This study aimed at identifying fish allergens of salmon and grass carp and evaluating the sensitization pattern in fish allergic subjects from two distinct populations in Asia. Methods: One hundred and three fish allergic subjects were recruited from Hong Kong (67 subjects) and Japan (46 subjects). Western blot and mass spectrometry were used to identify allergens from salmon and grass carp. Fish allergens were purified and tested against 96 sera on ELISA to analyze patients’ sensitization pattern. The protein profiles of salmon meat prepared under different cooking methods until core temperature reached 80 °C were evaluated by SDS-PAGE and mass spectrometry. Results: Three common allergens between salmon and grass carp, namely enolase, glycerldehyde-3-phosphate dehydrogenase (GAPDH) and parvalbumin, and two salmon-specific allergens collagen and aldolase were identified. Parvalbumin was the major allergen for both fishes showing an overall sensitization rate of 74.7%, followed by collagen (38.9%), aldolase (38.5%) and enolase (17.8%). Japanese subjects showed more diverse allergen sensitization pattern and more frequent IgE-binding to heat-labile salmon allergens. Compared with steaming and boiling, cooking by baking and frying retained more fish proteins inclusive of heat-labile allergens. Conclusions: Fish allergic patients from different Asian populations show varying fish allergen sensitization profiles. The relevant extracts and components for diagnosis are population-dependent but parvalbumin and collagen are important biomarkers. Cooking methods modify allergen composition of salmon and appear to influence patients’ allergic manifestations.