Closely related Quercus species generally exhibit low levels of genetic differentiation despite their ecological and morphological differences. However, at a few so-called ‘outlier’ loci they seem to remain genetically distinct. Isocitrate dehydrogenases (IDH) are key enzymes involved in the metabolic pathway of the citrate cycle. IDH has also been characterised as an ‘outlier’ marker, significantly differentiating the closely related Q. robur and Q. petraea with the isozyme technique. This ability to differentiate the species was tested here at molecular level: 13 single nucleotide polymorphism (SNP) markers were identified and developed within a NADP+-specific IDH gene in Quercus spp. and applied as molecular markers in a four species mixed oak forest in eastern Europe, where Q. robur, Q. petraea, Q. pubescens and Q. frainetto naturally co-exist. From the 13 developed SNPs, three groups were formed: non-synonymous, synonymous and non-coding SNPs. The levels of total gene diversity were moderate for all species investigated. The non-synonymous SNPs showed lower levels of gene diversity. Overall, the four closely related Quercus spp. were significantly differentiated (except Q. petraea with Q. frainetto). Analysis of non-random association of alleles revealed no clear physical clustering of the SNP sites in significant linkage disequilibrium (LD). However, separate LD analysis for each species showed a lower number of sites in significant LD for Q. robur than for the other species, possibly reflecting the history of the species in this specific geographical site and less efficient recombination effect due to the larger effective population size of Q. robur. Eleven statistically significant associations were found between seven SNPs and morphological traits that are commonly used to differentiate oak species.