The efficiency of the excitation capture by open Photosystem II (PSII) reaction centres was measured by the Fv/Fm ratios in a collection of winter and spring oats in order to assess the effects of hardening and freezing on the functionality of PSII and also the suitability of a chlorophyll fluorescence-based method to screen oat cultivars for frost tolerance. A significant reversible decrease in Fv/Fm was found in all genotypes during acclimation to low, non-freezing temperatures. Fv/Fm analysis appears to be an attractive test for the evaluation of frost tolerance in oats, being rapid, non-invasive and capable of monitoring a trait related to a crucial stage in the acquisition of frost tolerance. It is more sensitive and precise than other standard methods and highly correlated with field-evaluated frost damage. The measurements made during recovery 1 or 2 days after stress when the visual symptoms are not yet expressed, were especially advantageous because of the large variability in genotype response. The r-values (close to 0.8) were reduced due to the non-standard behaviour of the winter cultivar ‘Aintree’. The cold acclimation response of this genotype has been analysed in detail and the limits of artificial freezing tests are discussed.