Berberis microphylla, commonly known as calafate, is one of the most promising species of Chilean Patagonia to be domesticated, due to its anthocyanin-rich berries. The main aim of this study was to understand the genetic structure of the wild populations of B. microphylla in the main regions where it grows and its relationship with phenolic secondary metabolite profiles. Ripe berry samples and leaves were collected from the Aysén and Magallanes regions. Genetic analyses were carried out using 18 microsatellite markers. Phenolic compounds were extracted from the ripe fruits and were quantified using high-performance liquid chromatography (HPLC). Their antioxidant capacity was determined according to the Trolox equivalent antioxidant capacity (TEAC) assay. Total phenols were measured as their absorbance at a wavelength of 280 nm. Both the genetic and chemometric data were explored using unsupervised and supervised methods. The genetic markers suggest the existence of three groups, two of them corresponding to the Aysén and Magallanes samples, and the third corresponding to Chile Chico (a district in Aysén), which was the most divergent of the three. Similar results were observed in the phenolic profile obtained with chemometric analysis, with the same samples forming a separate third group. The differentiation achieved using the genetic and chemical data may be the result of intrinsic genetic differences, environmental effects on fruit maturity, or the sum of both factors. These are all points to consider in the domestication of this valuable species by selecting individuals with desirable traits and contrasting phenotypes.