We describe a scenario of plant speciation across a relict forest archipelago in South Africa involving Pleistocene habitat expansion-contraction cycles, dispersal and adaptation to lower temperatures. This is the first population level study using molecular data in South African forests and has significant implications for conservation efforts in this area. Populations of the mesophytic forest floor herbs Streptocarpus primulifolius sensu lato and Streptocarpus rexii were sampled throughout their range in the naturally fragmented forests of eastern South Africa in order to investigate population genetic and phylogenetic patterns within the species complex, using nuclear microsatellites, nuclear ribosomal ITS (internal transcribed spacer) sequences and chloroplast genome sequences. S. primulifolius harbours high levels of genetic diversity at both the nuclear (mean HE = 0.50) and the chloroplast level (each population fixed for a unique haplotype). This is consistent with populations of these coastal species being Pleistocene relicts. In contrast, populations of S. rexii in cooler habitats at higher altitudes and lower latitudes harbour little or no nuclear genetic diversity (mean HE = 0.09) and most share a common chloroplast haplotype. The split of S. rexii from populations intermediate between the two species (S. cf. primulifolius) occurred between 0 and 0.44 million years ago according to the calibrated ITS phylogeny of the taxa. The low genetic diversity and homogeneity of S. rexii is congruent with this species having reached its current range during the Holocene. We found no evidence of monophyly of any of the taxa in this study, which we consider a consequence of recent evolution in a fragmented habitat.