Begomoviruses are important crop viral disease agents, and they are transmitted by whiteflies of the Bemisia tabaci complex. Although the transmission of begomoviruses by whiteflies has been studied for many years, the mechanisms governing differential transmission of begomoviruses by different species of the Bemisia tabaci complex remain largely unknown. Here we firstly compared the transmission efficiency of tobacco curly shoot virus (TbCSV) by four species of the B. tabaci complex and found that Asia II 1 transmitted this virus with the highest efficiency, whereas MEAM1 transmitted it with the lowest. Next, by performing quantitative analysis of virus and immune-fluorescence detection, we found that the efficiency of TbCSV to cross the midgut wall was higher in Asia II 1 than in MEAM1. Finally, we set the quantities of virions in the haemolymph to the same level in Asia II 1 and MEAM1 via injection and then compared their capacity in TbCSV transmission, and found that the difference in TbCSV transmission between them became smaller. Taken together, our findings suggest that the efficiency of a begomovirus to cross the midgut wall of a whitefly to reach the vector's haemolymph plays a significant role in determining transmission of the virus.