Understanding the spatiotemporal distribution of genetic variation and the ways in which this distribution is connected to the ecological context of natural populations is fundamental for understanding the nature and mode of intraspecific and, ultimately, interspecific differentiation. The Petunia axillaris complex is endemic to the grasslands of southern South America and includes three subspecies: P. a. axillaris, P. a. parodii and P. a. subandina. These subspecies are traditionally delimited based on both geography and floral morphology, although the latter is highly variable. Here, we determined the patterns of genetic (nuclear and cpDNA), morphological and ecological (bioclimatic) variation of a large number of P. axillaris populations and found that they are mostly coincident with subspecies delimitation. The nuclear data suggest that the subspecies are likely independent evolutionary units, and their morphological differences may be associated with local adaptations to diverse climatic and/or edaphic conditions and population isolation. The demographic dynamics over time estimated by skyline plot analyses showed different patterns for each subspecies in the last 100 000 years, which is compatible with a divergence time between 35 000 and 107 000 years ago between P. a. axillaris and P. a. parodii, as estimated with the IMa program. Coalescent simulation tests using Approximate Bayesian Computation do not support previous suggestions of extensive gene flow between P. a. axillaris and P. a. parodii in their contact zone.