The aim of this study was to evaluate the effect of fast and slow freezing and frozen storage on the metabolite content of six blackberry cultivars. The content of metabolites determined with HPLC RI/PDA-MS in stored blackberries was compared with the initial content of the fruit. During frozen storage of fruits a loss of vitamin C up to 80% has been recorded along with changes of color values, which shifted to blue and yellow hues. The color changes were accompanied with increased pH levels and content of anthocyanins. Most of the phenolic groups, sugars, and organic acids showed a better extraction after storage, especially in the slow freezing treatment due to a higher degree of tissue damage by freezing. The ‘Thornless Evergreen’ cultivar was especially rich in sugars, vitamin C, and phenolic compounds, but the highest levels of anthocyanins were determined in ‘Loch Ness’ cultivar.