Aim of the study: To evaluate the adaptive genetic variability of chestnut (Castanea sativa Mill.) populations from southern Spain in relation to bud burst and water stress. Area of study: Andalusia (southern Spain) where many chestnut groves were progressively abandoned and have become ‘naturalized’. Material and methods: A total of 126 chestnut trees from eight populations were assessed by means of nine genic microsatellite loci (expressed sequence tag simple sequence repeat markers) related to bud burst and water stress. Main results: Significant differences in genetic diversity were detected within and among populations, not found with neutral microsatellite markers. The structure analysis indicated the presence of two different gene pools. Research highlights: These results could contribute to the development of conservation strategies for this species in southern areas exposed to the effects of climate change. The genetic diversity of these populations could be useful in minimizing this risk and other predictable factors related to global change.