Nicotiana alata is resistant to Tomato spotted wilt virus (TSWV) and of great value in breeding. However, hybrid sterility constrains the application of interspecific genetic resources. Previously, we obtained interspecific hybrids between a cytoplasmic male sterility (CMS) line of Nicotiana tabacum and N. alata, some of which were pollen sterile. In the present research, we studied the cytological abnormalities during pollen development in sterile hybrids (F₁-D) by comparing pollen development with that in fertile hybrids (F₁-S) from the same cross. Transmission electron microscopy and DiI staining showed that the membrane structures of microspores and pollen in F₁-D sterile hybrids were impaired. Carbol fuchsin staining revealed that cytomixis, chromosome loss and asymmetric callose wall formation occurred with high frequency in the microsporocytes and microspores of the sterile hybrids. The cytoplasm and nucleus were lost in the microspores and pollen of sterile hybrids, leading to mature pollen grains that were vacuous and collapsed in the aperture region. In addition, delayed tapetum degradation was detected in the anther of sterile hybrids, and sporopollenin was deposited in the aperture region. Impaired membrane structures of microspores and pollen in F₁-D sterile hybrids affected the integrity of the cells, and might be associated with chromosome, nuclear and cytoplasm loss, vacuous pollen, and sterility in F₁-D hybrids. Abnormal tapetum degradation in the anther and irregular sporopollenin deposition in the pollen wall of the F₁-D sterile hybrids might also be related to the pollen sterility. This study deepens our understanding of the cytological mechanisms of hybrid sterility, and may facilitate the application of TSWV-resistant resources in cultivated Nicotiana species through hybrid fertility restoration and backcross breeding.