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The area occupied by tropical forests in South America has continually experienced shrinkage since the Miocene, with the formation of the Dry Diagonal, which is composed of seasonally dry tropical forests and savannas (Caatinga, Cerrado, and Chaco). Here, we contribute to the understanding of the establishment of the Dry Diagonal by testing the hypotheses of “ancestral fragmentation” and of “colonization” to explain the current distribution of species in rocky humid formations of Cerrado, a Neotropical savanna in Brazil. Populations of Mandirola hirsuta (Gesneriaceae) were sampled in 25 localities; these cover the species’ entire geographical distribution, and the plastid intergenic regions psbA-trnH and trnQ-5′-rps16 were sequenced. The findings show that intrapopulation genetic variability was low, whereas interpopulation variability was high. We detected seven genetic groups that were geographically well delimited, and no signs of recent population expansion were observed. Divergence between the seven main lineages took place between the late Miocene and the Pliocene periods. In Approximate Bayesian Computation, the fragmentation scenario had a significantly higher probability than the colonization scenario. Results support the hypothesis of a former broader distribution for the humid vegetation, which was fragmented with the expansion of savannas; results also indicate population differentiation promoted by a rapid expansion of the Dry Diagonal.