An improved in vitro pollen germination method was developed for avocado (Persea americana Mill.). The effect of different concentrations of sucrose, polyethylene glycol (PEG), Mg and Ca on pollen germination was evaluated in order to determine the optimal pollen germination medium, i.e. that maximizing the percentage of pollen germination and minimizing the percentage of bursted pollen grains. Once the germination medium was optimized we used it to study the effect of temperature on in vitro pollen germination and tube growth in different cultivars from the three botanical varieties of avocado, that differ in their adaptation to environmental conditions. Significant differences in percentage of pollen germination and in pollen tube growth were observed among cultivars. These results could have implications not only for optimizing pollen management in avocado but also to select the best pollinizers for a particular cultivar.