Abstract: To explore the palynological characteristics of Nicotiana and its taxonomic significance, we examined pollen morphology of 36 wild species and 4 cultivated varieties using scanning electron microscopy, and investigated their taxonomic relationships through cluster analysis. The results revealed that all 40 accessions exhibited monad, isopolar pollen grains with tricolporate or tetracolporate apertures, displaying spherical to prolate shapes P/E=1.66(0.99–2.16) and measuring 39.96 (26.55–58.08) μm × 24.28 (17.83–33.79) μm. Seven exine ornamentation types were identified, including verrucate, verrucate-rugulate, rugulate, rugulate-striate, striate, striate-reticulate, and reticulate, among which the striate type was the most prevalent, and most species exhibited exine perforations. Pollen size and exine ornamentation were identified as the key diagnostic characteristics for Nicotiana pollen differentiation. Cluster analysis based on characteristic values classified the 40 taxa into three groups at a similarity coefficient of 15.73. The clustering results of six sections(Nicotiana sect. Nicotiana, Nicotiana sect. Paniculatae, Nicotiana sect. Petunioides, Nicotiana sect. Repandae, Nicotiana sect. Tomentosae, and Nicotiana sect. Trigonophyllae) were consistent with the Knapp taxonomic system. The palynological results provide significant corroborative evidence for the taxonomic study of Nicotiana, with the classification results generally supporting findings from molecular systematic research.