Canker stain, caused by the fungus Ceratocystis platani, is a destructive disease in Platanus spp. It has been recently proved that resistant accessions can be produced and grown in Europe. However, additional resistant genotypes are still needed in order to avoid the onset of virulent pathogen strains favoured by the selection pressures exerted by genetically homogeneous resistant plane tree plantings. In this study we present the results of two parallel experiments performed on 975 accessions of P. × acerifolia seedlings and P. × acerifolia clones derived by cutting propagation from mature trees grown in the urban environments. The selection process was based on inoculation with C. platani and yielded 13 accessions that showed different types of resistant reactions and survived in a stable manner thoughout the period of observation. Selected accessions were characterised by sequencing the rDNA-ITS region and by developing PCR procedures capable of detecting P. orientalis and P. occidentalis LEAFY homoeologues. These molecular analyses enabled us to confirm the identification of the species, its hybrid origin and to assess an evident genetic variability among the accessions, which therefore have to be considered as different genotypes.