The B-biotype of Bemisia tabaci (Homoptera: Aleyrodidae) has become extremely resistant to commonly used insecticides in China. To further explore the mechanisms of resistance to diafenthiuron, the diafenthiuron induction profiles of carboxylesterase (COE1), glutathione S-transferase (GST) and seven cytochrome P450 genes in both resistant (R-DfWf) and susceptible (S-Lab) strains were characterized. The detoxification genes GST, CYP6CX4, CYP6DW3, CYP6DZ6 and CYP9F, which are known to be constitutively over-expressed in the R-DfWf strain, were significantly upregulated in R-DfWf and S-Lab strains exposed to diafenthiuron at LC50 compared with their levels in strains treated with distilled water (controls); however, CYP6CX1, another detoxification gene, was not upregulated. The upregulation was more pronounced in the R-DfWf strain than in the S-Lab strain exposed to different concentrations of diafenthiuron (LC10 or LC50). Interestingly, COE1, CYP6CM1 and CYP6A, which are not constitutively over-expressed in the R-DfWf strain, were all significantly upregulated after exposure to diafenthiuron. Similarly, significant differences in the expression of these detoxification genes, with the exception of CYP6CM1 in the S-Lab strain, were also observed after exposure to diafenthiuron. However, the induction of CYP6A and COE1 was more pronounced in the S-Lab strain than in the R-DfWf strain after treatment with diafenthiuron at both concentrations, indicating that diafenthiuron induction of CYP6CM1 is specific to the R-DfWf strain, while diafenthiuron induction of the other genes is common to both the R-DfWf and S-Lab strains. These results demonstrate that multiple detoxification genes are co-upregulated in the R-DfWf strain through both constitutive over-expression and induction mechanisms. This knowledge will be useful for rational selection of insecticides for use in resistance management and control of this species.