Cold stress in citrus fields is threatening fruit production and the establishment of new fields in the north of Florida. Losses in the photosynthetic capacity of citrus plants under low temperatures result from stomatic and non-stomatic limitations derived from structural damage in cell membranes. The present study was conducted to gain insight into how the photosynthetic machinery of citrus leaves is affected by freezing temperatures, using the method of OJIP fluorescence transients. Two-year-old citrus plants were transferred to the growth chamber and posteriorly submitted to a progressive temperature reduction from 22 ^oC to reach -6 ^oC. The plants were kept at -6 ^oC by 1.5 h. The OJIP test was measured on leaves (n = 6) that were darkness-adapted using a portable LI-6800 fluorometer. A light pulse of 1 μmol photons m⁻² s⁻¹ for 50 ms was applied to determine the minimum fluorescence (F_o), followed immediately by a light saturation pulse of 3000 μmol photons m⁻² s⁻¹ for 1 s to determine the maximum fluorescence (Fm), the maximum photosynthetic quantum yield of PSII (F_v/F_m), and the actual quantum yield of PSII (photosystem II) electron transport (Φ_PSII). Freezing stress caused a significant reduction in F_o, F_m, F_v/F_m, and ΦPSII parameters, confirming structural and functional damage in the photosynthetic machinery of citrus leaves. On the other hand, OJIP curves showed reductions in the fluorescence levels in the J-I and I-P stages, suggesting limitations in the reduction status of electron carriers (J-I stage) and dysfunctionality in the PSI (photosystem I) because of a low electron flow (I-P stage). Our data demonstrated the negative impact of the freezing stress at specific points of the electron transport chain of photosynthesis. In addition, the OJIP test is a powerful tool to characterize photosynthetic damage and its promising use in high-throughput plant phenotyping programs, which are focused on the selection of citrus plants with high basal tolerance to cold stress.