AbstractBACKGROUNDHydrolysis of the fruit phenolic glucosides occurring during the oil extraction process is the main biochemical reaction affecting the biosynthesis and accumulation of secoiridoid compounds in virgin olive oil. An integrated approach at the molecular, biochemical, and metabolic level was used to study the olive β‐glucosidase gene family in seven olive cultivars selected by their different phenolic profiles.RESULTSEight β‐glucosidase genes have been identified by in silico analysis of an olive transcriptome. Their expression levels were analyzed by reverse transcription quantitative polymerase chain reaction in olive fruits at different ripening stages: I, green fruits, 16–19 weeks after flowering (WAF); II, yellow–green fruits, 22–25 WAF; III, turning fruits, 28–31 WAF; and IV, fully ripe fruits, 35–40 WAF. Gene expression was compared with the level of β‐glucosidase activity in the fruit and with the phenolic composition of fruits and oils from different olive cultivars. Phylogenetic analysis of the encoded proteins and differences found among the β‐glucosidase genes based on Gene Ontology enrichment analysis data suggests maximum involvement of two genes, OeBGLU1A and OeBGLU1B, in the phenolic composition of virgin olive oil. Positive correlation coefficients were found within each olive cultivar between OeBGLU1A and OeBGLU1B gene expression data and the phenolic content of the oil.CONCLUSIONThe results obtained suggest that the expression pattern of specific β‐glucosidase genes may be an accurate predictor for the phenolic content of virgin olive oil that could be used in olive breeding programs. © 2021 Society of Chemical Industry.