Hydroxytyrosol derivatives are the most important phenolic components in virgin olive oil due to their well-demonstrated biological activities. In this regard, two phenyl acetaldehyde reductase genes, OePAR1.1 and OePAR1.2, involved in hydroxytyrosol synthesis, have been identified from an olive transcriptome. Both genes were synthesized and expressed in Escherichia coli, and their encoded proteins were purified. The recombinant enzymes display high substrate specificity for 2,4-dihydroxyphenylacetaldehyde (3,4-DHPAA) to form hydroxytyrosol. The reaction catalyzed by OePAR constitutes the second, and last, biochemical step in the formation of hydroxytyrosol from the amino acid L-3,4-dihydroxyphenylalanine (L-DOPA) in olive. OePAR1.1 and OePAR1.2 enzymes exhibit high thermal stability, similar pH optima (pH 6.5), and high affinity for 3,4-DHPAA (apparent Km 0.6 and 0.8 µmol min-1 mg-1, respectively). However, OePAR1.2 exhibited higher specific activity and higher expression levels in all the olive cultivars under study. The expression analyses indicate that both OePAR1.1 and OePAR1.2 genes are temporally regulated in a cultivar-dependent manner. The information provided here could be of interest for olive breeding programs searching for new olive genotypes with the capacity to produce oils with higher levels of hydroxytyrosol derivatives.