Pedunculate and sessile oaks (Quercus robur L.; Q. petraea [Matt] Liebl.) often coexist in mixed forest stands. However, species-specific investigations and forest management actions in such populations require reliable methods of identification of the species status of individuals. We investigated genetic diversity and species differentiation of adult and naturally established seedling cohorts in a mixed forest stand composed of Q. robur and Q. petraea, located in the Jamy Nature Reserve in north-central Poland. Using nineteen nuclear microsatellite loci and a model-based clustering approach as a tool for species delineation, we efficiently identified 105 and 60 adults, as well as 191 and 456 seedlings of pedunculate and sessile oaks, respectively. While the adult trees of both species were randomly distributed throughout the sample plot, the seedlings demonstrated significant spatial clustering, which was particularly evident for Q. petraea. The two oak species exhibited similar levels of genetic diversity in adult and offspring cohorts. Inbreeding was found to be low and significant only at the stage of seedlings. The estimates of effective population size were higher for Q. robur than Q. petraea, despite the overall greater reproductive success of the later one. There was a significant level of differentiation between the studied oak species, as measured by Fst coefficient (0.084 – adults; 0.099 – seedlings). The results on genetic diversity and species differentiation obtained in the studied indigenous near-natural stand of Q. robur and Q. petraea could be considered as a reference for other population genetic studies of oaks.