Rise of temperatures and shortening of available water as result of predicted climate change will impose significant pressure on long-lived forest tree species. Inter- as well as intraspecific diversity is the key element of a plants potential to adapt to a changing environment and tolerance towards drought stress. In the present study, we have used Roche 454 sequencing and developed a bioinformatic pipeline to process multiplexed tagged amplicons in order to identify single nucleotide polymorphisms and allelic sequences of candidate genes related to drought/osmotic stress from pedunculate oak (Quercus robur) and sessile oak (Q. petraea) individuals. Out of these, eight genes of 336 oak individuals growing in Austria have been detected with a total number of 158 polymorphic sites. Nucleotide diversity, correlations to environmental conditions, and deviations from standard neutral models were examined, baseline genetic parameters were calculated and population structure was investigated using differentiation indices and Bayesian clustering. Significant differentiation and strong correlations between the local temperature-precipitation regime and the allele frequencies point towards a higher adaptive potential of Q. petraea under arid conditions. These results can serve as basis for future studies in larger geographic scale and may be used for the development of molecular markers for drought tolerance.