ABSTRACT Tropospheric ozone (O3) is a phytotoxic air pollutant and the O3-induced visible foliar injury (O3 VFI) is a biomarker. A recently developed Free-air O3 eXposure (FO3X) is a promising facility to verify field-observed “O3-like” VFIs and to establish a flux-based threshold for the O3 VFI onset. The present study compared O3-like VFI registered in the southern European forest sites with actual O3 VFI observed in a FO3X experiment. The O3-like VFIs were evaluated by eye in forests and thus it was subjective. According to the imaging analysis, we firstly demonstrated that major parts of the colors were similar in the field and the FO3X. The color pallets for O3 VFI was species-specific and considered a advanced tool for the O3 VFI diagnosis. In addition, we calculated a flux-based threshold for the O3 VFI onset at the FO3X based on a Phytotoxic Ozone Dose (POD1), which ranged from 4.9 to 18.1 mmol m−2 POD1. This FO3X-derived threshold partly explained but did not necessarily match with the observation for several tree species in actual forests. The multivariate analysis showed that O3 VFI was decreased by the presence of various species and suggested the importance of continuous monitoring activities in the field for the further analysis.