Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is a species complex that is one of the most devastating agricultural pests worldwide and affects a broad range of food, fiber, and ornamental crops. It is multivoltine and polyphagous, and vectors economically important plant viruses including those belonging to the family Geminiviridae. Therefore, understanding genetic variation among vector populations is important for its management. In order to gain insight into whitefly genotypes occurring on ash gourd, Benincasa hispida (Thunberg) Cogniaux, whitefly samples were collected from 18 locations in Tamil Nadu, and their mitochondrial cytochrome oxidase subunit I (mtCOI) genes were sequenced. Sequences generated in this study, when compared with sequences from the mtCOI data set, revealed that the whitefly populations TN1 Ja, TN2 Po, TN3 Ud, TN4 Ma, TN5 Ga, TN6 Co, TN9 Td, TN10 Gp, and TN11 El belong to Asia I genotype. Contrastingly, the populations TN7 On and TN8 Er were identified as Asia II 6 genotype. Interestingly, more than one genetic group was found coexisting in the same field.