Blueberry (Vaccinium corymbosum L.) is a widely consumed fruit and a rich source of bioactive compounds, namely, the polyphenol class of anthocyanins. Little information is available about the influence of internal (genetic and developmental) and external (environmental) factors on the levels of phenolic metabolites in blueberry fruit. In light of this consideration, total polyphenolic and flavonoid content, anthocyanin accumulation and composition were evaluated in cv. “Duke” and “Brigitta” grown at two different altitudes in Valtellina, a valley of the Alps in Northern Italy. During berry ripening, there is a developmentally coordinated shift from cyanidin-type, di-substituted anthocyanins toward delphinidin-based, tri-substituted pigments. At the lower altitude location, higher temperatures, not exceeding optimum, resulted in a more quickly berry developmental pattern and in higher anthocyanin concentrations in the early phases of ripening. At later stages of ripening, berries of both cultivars at higher altitude compensate for these initial temperature effects, and no differences were recorded in ripe fruit grown in the two locations. We conclude that anthocyanin accumulation is strongly regulated by development and genotype, and the environmental factors, associated to the altitude gradient, exert in the trial conditions only a fine-tuning influence. Fruits reach the full-ripening stage simultaneously at both sites because the initial gap in pigment levels is counterbalanced at the higher altitude by a faster rate of accumulation at later phases of the ripening process.