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The phylogenetic relationship between the whitefly Bemisia afer (Priesner & Hosny) (Hemiptera: Aleyrodidae) from China and other populations among the world were analyzed based on the mitochondrial cytochrome oxidase I (mtCOI) gene. Phylogenetic analysis of mtCOI sequences and those of reference B. afer sequences showed that the populations of the species could be separated into 5 clades (I–V). There were at least two clades of the species from China (IV and V). These data suggested that B. afer might be a species complex. The Chinese B. afer populations were most divergent with B. afer from the United Kingdom and African countries. The distance between the Chinese B. afer (IV and V) and clades I, II, and III is more than 32%, while the distance among clades I, II, III is lower than 7.7%. A new set of primers specific to B. afer was designed to amplify a region of approximately 400 bp to discriminate B. afer from other Bemisia species in China based on mtCOI sequences.