The Guianas have often been proposed as a forest refugium; however, this view has received little testing. Studies of population genetics of forest taxa suggest that the central part of French Guiana remained forested, while the southern part (currently forested) may have harboured more open vegetation. Insights into the population structure of species restricted to non-forested habitats can help test this hypothesis. Using six microsatellite loci, we investigated the population genetics of French Guianan accessions of Manihot esculenta ssp. flabellifolia, a taxon restricted to coastal savannas and to rocky outcrops in the densely forested inland. Coastal populations were highly differentiated from one another, and our data suggest a recent colonization of these savannas by M. esculenta ssp. flabellifolia in a west-to-east process. Coastal populations were strongly differentiated from inselberg populations, consistent with an ancient separation of these two groups, with no or low subsequent gene flow. This supports the hypothesis that the central part of the region may have remained forested since the Last Glacial Maximum, impeding the establishment of Manihot. Contrary to coastal populations, inselberg Manihot populations were strikingly homogeneous at a broad spatial scale. This suggests they were connected until recently, either by a large continuous savanna area or by smaller, temporary disturbed areas shifting in space.