In the last 30 years the incidence of kiwifruit allergy has increased with the three major allergenic proteins being identified as actinidin, kiwellin, and thaumatin-like protein (TLP). We report wide variation in the levels of actinidin and TLP in 15 kiwifruit varieties from the four most widely cultivated Actinidia species. Acidic and basic isoforms of actinidin were identified in Actinidia deliciosa 'Hayward' and Actinidia arguta 'Hortgem Tahi', while only a basic isoform of actinidin was identified in Actinidia chinensis 'Hort16A'. One isoform each of kiwellin and TLP were identified in ripe fruit. The cysteine protease activity of actinidin correlated with protein levels in all species except A. arguta. Protein modeling suggested that modifications to the S2 binding pocket influenced substrate specificity of the A. arguta enzyme. Our results indicate that care is necessary when extrapolating allergenicity results from single varieties to others within the same and between different Actinidia species.