Abstract: Meiosis plays an important role in gamete formation of plants during sexual reproduction. Meiosis is also a research hotspot in reproductive biology. To promote the progress of reproductive biology research in Populus canescens, meiosis and microtubule cytoskeleton of pollen mother cells (PMCs) were conducted in P. canescens by indirect immunofluorescence microscopy and aceto-carmine staining. The results showed that: 1) colors of anther could be considered as a morphological marker to discriminate meiotic stages of PMCs. With PMCs developing from leptotene to tetrad, the colors of anthers varied from peak green, yellow green, slight red, light red, fresh red to crimson. 2) Lagging chromosomes at telophase Ⅰ connected with centromeric microtubule was observed in PMCs. Owing to the balanced pull of centromeric microtubule, chromosomes did not move to the polar of PMCs and stayed at the center of cell plate, resulting in the loss of chromosomes. Therefore, it was inferred that the mechanism of aborted pollen formation in P. canescens was the unbalance of chromosome numbers of male gametes. 3) Cytokinesis in P. canescens was of a typical simultaneous-type cytokinesis, which was regulated by nuclear-based radial microtubule systems. Cytokinesis was initiated at the edge of mother cells and centripetally prolonged, up to be four separate microspores. 4) Natural 2n pollens were observed in P. canescens. Parallel and fused spindles were considered to be responsible for the 2n pollen formation. In this study, the relationship between anther color and meiotic process of PMCs was firstly reported, and the cause of lagging chromosome formation and its aborted pollen mechanism were revealed, and the mechanism of natural 2n pollen formation was discussed. Our findings will provide a platform for making strategy of genetic improvement in P. canescens.