Little is known about genetic variation in members of the genus Aesculus (Hippocastanaceae), in particular A. flava (yellow buckeye) and A. glabra (Ohio buckeye). Here, three synthetic DNA probes (composed of tandemly repeated, core sequences) that reveal alleles at multiple variable-number tandem-repeat (VNTR) loci in these two species were used to investigate: 1) levels of genetic variation in one stand of A. flava and three isolated stands of A. glabra; 2) whether the stands of A. glabra are genetically differentiated from one another; 3) whether there has been selection for more heterozygous individuals through time in one stand each of A. flava and A. glabra; and 4) whether a possible genetic bottleneck had occurred during the formation of either species of Aesculus.