Phenolic glycosides, especially the salicylates, are important secondary metabolites in the bark of willows (Salix spp.). Because of their anti-inflammatory, analgesic, and fever-reducing properties, they are of particular interest to society. Compared to the fabrication of synthetic salicylacetylic acid, the commercial production of willow bark extracts with adequate amounts of salicylate is very difficult due to several biological and technical reasons. Therefore, one of the objectives was to identify salicylate-rich clones from three species, Salix daphnoides , Salix purpurea , and Salix pentandra , with potentially high amounts of phenolic glycosides. Three hundred different Salix clones were collected, and the chemical profiles of their bark were analyzed by HPLC. Overall, S. daphnoides clones showed the highest phenolic glycoside contents, followed by S. purpurea and S. pentandra. Second, seasonal changes of secondary compounds in willow bark were analyzed to determine the optimal harvesting time. The phenolic glycoside levels decreased over the growing season, with highest contents detected during plant dormancy. The effects of different cultivation conditions were also examined, and none of these treatments were found to have a significant effect on the phenolic glycoside content in willow bark. Biomass accumulation in the clones with grass competition was significantly lower than in the other three treatments.