<p><strong>Objective: </strong>To observe gender specific seasonal variation of two phenolic glycosides (PG’s) (populin and salicin) in <em>Populus ciliata</em> male and female trees.</p><p><strong>Methods: </strong>Plant material (bark) was collected from male and female trees throughout the year. The content of salicin and populin was measured using HPLC. Because of the lack of populin in the market, the standard compound was synthesized in the laboratory from salicin following standard procedure. Confirmation and characterization of synthesized populin were done using <sup>1</sup>H NMR and <sup>13</sup>C NMR. TLC and LC-MS of methanolic extract were performed to observe the presence of populin and salicin in the plant bark.</p><p><strong>Results: </strong>TLC showed the presence of populin and salicin in crude plant extract at Rf value 0.84 and 0.52, respectively. The results of monthly variation showed a consistent pattern of two PG’s for both the sexes. However, salicin content was observed highest compared to populin content. Whereas female trees were observed with low content of PG’s compared to male trees. The maximum content of salicin and populin was observed in the flowering season. LC-MS of bark methanolic extract confirmed the presence of a significantly larger peak, which was identified as a cinnamoyl-salicin peak at 463.</p><p><strong>Conclusion: </strong>The study confirms the highest content of PG’s in the flowering season. Additionally, LC-MS study concluded a new compound cinnamoyl-salicin (M<sup>+</sup>at 463) which is reported first time to the best of author’s knowledge. It seems that it could be the denaturation product of 2'-O-cinnamoyl salicortin and can be further explored for biological applications.</p>