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Ilex guayusa Loes. is a shrub native to the Neotropics, traditionally consumed as an infusion. Despite its cultural value and extensive use, genetic research remains scarce. This study examined the genetic and clonal diversity of guayusa in three different Ecuadorian Amazon regions using 17 species-specific SSR markers. The results obtained suggest a moderately low degree of genetic diversity (He = 0.396). Among the 88 samples studied, 71 unique multilocus genotypes (MLGs) were identified, demonstrating a high genotypic diversity. A Discriminant Analysis of Principal Components (DAPC) revealed the existence of two genetic clusters. We propose that a model of isolation-by-environment (IBE) could explain the genetic differentiation between these clusters, with the main variables shaping the population’s genetic structure being temperature seasonality (SD × 100) (Bio 4) and isothermality ×100 (Bio 3). Nonetheless, we cannot dismiss the possibility that human activities could also impact the genetic diversity and distribution of this species. This study gives a first glance at the genetic diversity of I. guayusa in the Ecuadorian Amazon. It could assist in developing successful conservation and breeding programs, which could promote the economic growth of local communities and reinforce the value of ancestral knowledge.