INTRODUCTION: In China, Typhae Pollen (TP) has been extensively utilized as a medicinal product, which is the dried pollen from the male inflorescences of Typha angustifolia L. (TA), Typha orientalis Presl (TO), or species within the same genus. However, existing methods for differentiating the botanical origin and controlling the quality of TP are confusing, unreliable, and unsystematic. OBJECTIVES: This study aims to elucidate the microstructural similarities and differences between pollen grains of the two species and to develop a comprehensive method for the qualitative and quantitative analysis of multiple flavonoids and phenolic acids in TP. METHODOLOGY: Scanning electron microscopy (SEM) was employed to examine the dried pollen grains. A comprehensive analytical approach was developed using ultra-high performance liquid chromatography with diode array detection and quadrupole-time-of-flight mass spectrometry to quantitatively and qualitatively analyze 17 flavonoids and phenolic acids. Discrepancies in constituents were further explored using partial least squares discrimination analysis and hierarchical clustering analysis. RESULTS: Microscopic examination revealed distinct differences between the pollen grains of the two species, which could be distinguished using SEM. The analytical methods established for these 17 constituents-comprising 13 flavonoids and 4 phenolic acids-proved reliable and accurate. The flavonoid glycosides in TP could be categorized into three distinct groups. In TA, the average contents of constituents 1, 2, 3, 4, 8, and 12 were higher than in TO, whereas the reverse was true for constituents 7, 14, 16, and 17. Constituents 9, 10, and 13 were unique to TO. And constituent 14 should be chosen as a more appropriate quality indicator for TO. Chemometric techniques effectively differentiated between TA and TO. CONCLUSIONS: This study contributes to enhancing quality control and facilitating botanical origin identification of TP and provides an experimental foundation for improving its standards.