Morphological traits traditionally adopted to discriminate between Populus alba L. and P. tremula L. have frequently led to misclassification of their spontaneous hybrid P. · canescens Sm. Moreover, they may not be of any help in cases of spontaneous backcross phenomena. These limitations can be overcome by molecular markers, which are not environmentally influenced nor subjectively assessed. In this study, the effectiveness of amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers in species and hybrid discrimination was evaluated by analysing a set of reference samples of P. alba,P. tremula and P. · canescens. Species-specific and speciesindicative AFLPs, as well as diagnostic SSR alleles, were recorded in both P. alba and P. tremula reference samples. The results allowed a clear distinction between the two poplar species and their hybrid. Using these diagnostic markers, a natural population of P. alba trees sampled along the Ticino river basin in northern Italy was analysed, and P. · canescens individuals, intermingled with P. alba trees, were detected.