Abstract Central South America primarily comprises seasonally dry tropical domains, where a sharp rainy season defines phenological and yearly growth patterns. Here we examine if the genetic structure of the understorey ground-herb Chamaecostus subsessilis species complex, from South American seasonally dry forests, is defined by intrinsic contrasting environments of the Cerrado and southern Amazonia. We analysed a dataset of c. 20 kb of sequence data from targeted capture high-throughput sequencing using PCR-generated probes. We used an admixture model and population pairwise FST comparisons to interpret genetic structure. We also used a Bayesian coalescent approach with a relaxed clock to estimate divergence times between populations and haplotypes. Genetic structure reflects separate populations from Amazonia and from the Cerrado, whereas a haplotype-based phylogenetic tree provides further spatiotemporal evidence of this strong phylogeographical structure. Spatial arrangements among genetic lineages of the C. subsessilis species complex reveal that intraspecific evolution in Central South America seasonally dry understories is closely linked to environmental gradients.