The phenolic profiles of six varieties of avocado (Persea americana) grown in Sicily were investigated. The ultra-high-performance liquid chromatography-heated electrospray-mass spectrometry method was developed to determine qualitative and quantitative changes in fruits at two different ripening stage. Nineteen individual phenolic compounds were detected in avocado pulp extracts. Gallic acid, sinapinic acid, vanillin, p-coumaric acid, and gentisic acid were present only in ripe fruits. On the contrary, epicatechin decreased with fruit ripening, whereas protocatechuic acid, 4-hydroxybenzoic acid, chlorogenic acid, and benzoic acid were relatively stable or exhibited erratic changes with fruit ripening. The different avocado cultivars exhibited different phenolic profiles and total quantities and it was found that, among the tested cultivars, “Orotawa” avocados may provide the highest nutritional and health contribution to human diet. The qualitative and quantitative differences among cultivars and maturation stages by multivariate analysis allowed for the individuation of a set of phenolic compounds that have a great potential in the discrimination and identification of different genetic groups.