Species of Populus are widely distributed worldwide, playing a significant role in both ecology and economy. However, the lack of single-copy nuclear markers limits knowledge about the phylogeny and population genetics of this genus. In the present study, primer pairs of 15 single-copy nuclear markers were developed through bioinformatic methods based on complete genomic sequences of Populus trichocarpa and Salix arbutifolia. Twenty individuals of Populus davidiana Dode and Salix matsudana Koidz were used to evaluate the basic application of these markers with respect to marker length and diversity indices, respectively. The utility of single-copy nuclear markers is anticipated to facilitate further studies about the phylogeny, population genetics, and phylogeography of this genus, in addition to providing information about the evolutionary dynamics of Salicaceae.