The Populus L. genus includes tree species that are botanically grouped into several sections. This species successfully hybridizes both in the same section and among other sections. Poplar hybridization widely occurs in nature and in variety breeding. Therefore, the development of poplar species’ specific molecular markers is very important. The effective markers for trees of the Aigeiros Duby section have recently been developed using the polymorphism of 5S rDNA non-transcribed spacers (NTSs). In this article, 5S rDNA NTS-based markers were designed for several species of the Leuce Duby section. The alb9 marker amplifies one fragment with the DNA matrix of P. alba and P. × canescens (natural hybrid P. alba × P. tremula). The alb2 marker works the same way, except for the case with Populus bolleana. In this case, the amplification of three fragments was observed. The tremu1 marker amplification was detected with the DNA matrix of P. tremula and P. × canescens. Thus, the developed markers may be applied as a useful tool for P. alba, P. tremula, P. × canescens, and P. bolleana identification in various areas of plant science such as botany, dendrology, genetics of populations, variety breeding, etc.