Gaultheria pumila (L.f.) D.J. Middleton is a native shrub of Chile that produces edible berry fruits. This species is related to the cultivated Vaccinium species; for this reason it is currently called Chilean blueberry locally. Although G. pumila has important attributes, it has been largely ignored, and remains an unexplored genetic resource. This study investigates the genetic diversity to support the efforts to domesticate the species. Sampling was carried out in 11 sites collected from four Regions of Chile. In total, 160 individuals were collected and analyzed using a set of 10 simple sequence repeats (SSRs) markers. The average observed heterozygosity was Ho = 0.50, while the expected heterozygosity was He = 0.46. The fixation index (FIS) showed an average of -0.07, and the proportion of differentiation among populations (FST) was 0.11. The average level of polymorphic loci in all populations (PPL) was 96.97%. AMOVA showed that the genetic diversity among populations was very low (ΦPT = 6%). Significant correlations were found between genetic and geographic distance. Multivariate and Bayesian analyses identified two genetic groups. These results will be very useful to support the efforts to domesticate and increase the value of this species.