Low temperatures affect avocado plants and their response varies across genetic variants. The objective of this study was to assess cold stress of avocado cultivars based on visual, physiological, and biochemical criteria for evaluating freezing injury in these plants. The avocado cultivars 'Geada,' 'Fortuna,' 'Fuerte,' 'Quintal,' 'Margarida,' and 'Primavera' were subjected to temperatures of −2.5, −4.0, −5.0, and −6.0 °C for 1 h in a growth chamber. Their responses to cold stress were evaluated on the basis of visual damage score, photosynthetic rate, water pressure potential, protein content and enzymatic activity. Moreover, the experimental design was factorial and completely randomized with four replicates of each cultivar. An analysis of variance of the results was performed and the means were compared using Tukey's post-hoc test (p <0.05). Evaluations based on the plants water pressure potential, total protein content, catalase activity, photosynthetic rate, and visual damage score were efficient in estimating the effects of cold stress in avocado plants. Additionally, photosynthesis and visual damage score were significantly correlated in all evaluations, enabling classification of the cultivars in terms of response to cold stress. We found that Fuerte was the most tolerant cultivar followed by Geada. The Primavera was the most sensitive cultivar.