Anthocyanins protect plants against various biotic and abiotic stresses, and anthocyanin-rich foods exert benefits on human health due to their antioxidant activity. Nevertheless, little information is available on the influence of genetic and environmental factors on the anthocyanin content in olive fruits. Based on this consideration, the total anthocyanin content, the genes involved in anthocyanin biosynthesis, and three putative R2R3-MYB transcription factors were evaluated at different ripening stages in the drupes of the Carolea and Tondina cultivars, sampled at different altitudes in the Calabria region, Italy. During drupe ripening, the total anthocyanin content and the transcript levels of analyzed genes gradually increased. In line with the anthocyanin content, a different level of expression of anthocyanin structural genes was observed in ‘Carolea’ compared to ‘Tondina’, and in relation to the cultivation area. Furthermore, we identified Oeu050989.1 as a putative R2R3-MYB involved in the regulation of anthocyanin structural genes correlated with the environmental temperature change response. We conclude that anthocyanin accumulation is strongly regulated by development, genotype, and also by environmental factors such as temperature, associated with the altitude gradient. The obtained results contribute to reducing the current information gap regarding the molecular mechanisms on anthocyanin biosynthesis regulation related to the environmental conditions inOlea europaea.