Podocarpus sellowii (Podocarpaceae) is one of only a few gymnosperms native to Brazil and the sole species of the genus found in the northeastern region of that country. It has a very restricted distribution in this region, with only three known populations in highland forests (called Brejos de Altitude), which apparently have been isolated from each other since the Pleistocene. Due to this long-term isolation and the fact that these populations have few adult individuals and suffer great anthropogenic pressure, low genetic variability is expected, compromising their long-term viability. The present work assessed the genetic variability and structure of northeastern populations of P. sellowii to investigate the role of Pleistocene glaciations on the genetic relationships between them and to propose strategies for their conservation by analyzing the SSR and ISSR markers of adult and juvenile individuals. Low genetic diversity was found with both markers, associated with a high differentiation of the Brejo de Baturité population in relation to the others—suggesting their isolation at different points in time, probably during the Pleistocene. Actions directed towards increasing the genetic diversity of these populations will be needed, such as planting seedlings with high genetic variability—but the high degrees of differentiation observed between the populations must be taken into account.