One of the most devastating polyphagous pests in the world, Bemisia tabaci (Gennadius), causes significant damage to a variety of crops and has demonstrated the ability to develop resistance to different classes of insecticides rapidly. A novel pyridine azomethine derivative pyrifluquinazon exhibits exceptional insecticidal toxicity against B. tabaci by interrupting the function of chordotonal receptor neurons. Formulating resistance management strategies is crucial to ensure the long-term use of this insecticide for whitefly control; however, the characteristics and possible mechanisms of pyrifluquinazon resistance in B. tabaci remain unclear. By employing pyrifluquinazon selection for 22 successive generations, the pyrifluquinazon-resistant strain (PQZ-R) was generated from a laboratory-susceptible population of B. tabaci Asia I (Lab-WB) and exhibited 39.65-fold resistance to pyrifluquinazon. When the realized heritability (h2) of B. tabaci to pyrifluquinazon was assumed to be the laboratory-estimated value (h2 = 0.181) and the mortality was 70–90%, only 12.3–22.6 generations were expected to be required to obtain a 10-fold increase in pyrifluquinazon resistance. While there was no significant cross-resistance to cyantraniliprole, dinotefuran, flonicamid, flupyradifurone, pymetrozine, sulfoxaflor, or thiamethoxam, the PQZ-R strain displayed slight cross-resistance to afidopyropen (3.14-fold). Synergism tests indicated that piperonyl butoxide (PBO) inhibits (4.36-fold) pyrifluquinazon resistance in the PQZ-R strain. This impact may be attributed to enhanced detoxification (elevated 3.91-fold) mediated by cytochrome P450 monooxygenase (P450). Compared to Lab-WB, the PQZ-R strain exhibited no significant overexpression of the 13 published detoxification-related P450 genes from CYP303, CYP4, and CYP6 families in B. tabaci. The combined knowledge gained from this study will enable further investigations into the function of qualitative and quantitative variations in P450-encoded genes, leading to innovative approaches for efficiently managing B. tabaci.