The blueberry, a deciduous shrub in the Ericaceae family, is celebrated for its delightful flavor, sweetness, and abundance of anthocyanins and antioxidants, qualities that have garnered significant attention for their potential health benefits. Blueberries grown in diverse environments and exhibit varied anthocyanin profiles, often influenced by factors such as altitude and climate. Varietal groups worldwide have been bred and categorized based on their growth habits and specific cold requirements, particularly with southern highbush cultivars thriving in temperate climates, demonstrating tolerance to higher altitudes or cooler climates—a result of hybridizations involving various Vaccinium species. In the Colombian Andes, southern highbush blueberries thrive in unique high-altitude conditions, leading to exceptional quality due to the region’s cool climate and specific soil characteristics. In this context, this study aimed to chemically characterize and differentiate three southern highbush blueberry cultivars (i.e., ‘Biloxi,’ ‘Legacy’ and ‘Sharpblue’) cultivated in a Colombian Andean plateau and compare them to three commercially available highbush blueberries. This comprehensive evaluation involved examining total phenols, flavonoids, anthocyanin content, and DPPH· free-radical scavenging capacity, as well as conducting anthocyanin-targeted profiling via HPLC-DAD-HRMS. Through supervised multivariate analyses such as sPLS-DA, this study delved into the pattern recognition of those anthocyanins that could potentially serve as markers for quality and cultivar-related chemical trait determination. These findings locate blueberry-derived anthocyanins in a metabolic context and afford some insights into southern highbush blueberry cultivar differentiation to be used for further purposes.