Cotton leaf curl disease is one of the most significant constraints to the production of cotton. In the past decades our understanding of the begomoviruses (family Geminiviridae) causing the disease has improved, but little is known regarding transmission of these viruses by the different species of whiteflies in the Bemisia tabaci complex. We compared transmission efficiency of cotton leaf curl Multan virus (CLCuMuV), one of the major begomoviruses associated with cotton leaf curl disease, by four whitefly species, of which two are indigenous to Asia and two are invasive worldwide. Only the indigenous Asia II 1 species was able to transmit this virus with high efficiency. By quantifying the virus and using immunoflorescence assays, we found that the differential transmission was associated with the varying efficiency of CLCuMuV to cross the midgut of various whitefly species. Further, we verified the role of coat protein in the whitefly transmission of CLCuMuV. Based on a phylogenetic analysis of the virus coat proteins, we found that most begomoviruses associated with cotton leaf curl disease might share similar whitefly transmission characteristics. These findings advance our understanding of the nature of cotton leaf curl disease and provide information for the development of control and preventive strategies against this disease.