Bemisia tabaci adults from various host-plant species were collected from 31 regions across India. 266 B. tabaci samples were first screened by RAPD-PCR to examine molecular variability and to select individuals with different fingerprints. Host-plant and region of collection were then used to select 25 individuals for PCR amplification and sequencing of their partial mitochondrial cytochrome oxidase subunit one (mtCOI) genes. Pairwise comparisons with mtCOI consensus sequences showed that the majority of these samples had <3.5% sequence divergence from groups currently termed Asia I, Asia II-5, Asia II-7, and Asia II-8. The biotype-B B. tabaci from India clustered into the Middle East-Asia Minor 1 group. A new group of B. tabaci from Coimbatore, collected from pumpkin, was related most closely to the Asia I group (6.2% sequence divergence from the consensus Asia I sequence). To increase our understanding of the epidemiology of tomato leaf curl disease (ToLCD) and the key B. tabaci genetic groups involved in virus spread, the indigenous Asia I and the exotic biotype-B population from South India were used to carry out transmission experiments using Tomato leaf curl Bangalore virus (ToLCBV). The acquisition access periods (AAP), inoculation access periods (IAP), latent periods (LP), and ToLCBV transmission efficiencies of the two populations were compared and the biotype-B had the more efficient transmission characteristics. These results are discussed in relation to recent changes in the epidemiology of tomato leaf curl disease in South India.