Most almond cultivars are susceptible to negative temperatures, a limiting factor for almond expansion to regions with risks of spring frosts. As a consequence, the main objective of most almond breeding programmes is to select late- or very late-blooming genotypes, in order to avoid the periods of high frost risk. Flower and fruitlet tolerance to frosts has been only studied so far by observing the morphological damages produced by low temperatures. Thus, our objective was the evaluation of chlorophyll fluorescence (FC) to estimate the tolerance of 12 commercial almond cultivars from different origin to low temperatures. Flowers were maintained during 24 hours at different temperatures (0º, -1º, -2º and -3ºC), measuring FC afterwards. In general, the variable fluorescence (Fv) and the ratio Fv/Fm (being Fm the maximal fluorescence) decreased with time in all genotypes, although the decreasing rate depended on the genotype. The decrease of these parameters was slower in the cultivars tolerant to low temperatures, whereas the decrease was linear or/and sigmoid in the susceptible cultivars. In general, the classification of genotypes according to their frost tolerance level with this technique agreed with the published references. These results lay out that chlorophyll fluorescence is a promising technique (fast, quantitative, easy and non-destructive) to ascertain the tolerance of almond genotypes to frosts independently of their blooming time.