A mutation, leucine to isoleucine at position 925 (L925I) in the Ⅱ S 4-6 linker of the para type sodium channel protein from the Nanjing strain (B2biotype) of the tobacco whitefly, Bemisia tabaci (Gennadius) was identified to be associated with pyrethroid resistance. The DNA2based genotyping technique PASA (PCR amplification of specific allele) for rapid detection of the L925I mutation was established. The Nanjing strain collected from cottons in Nanjing in 2002 showed 772 fold resistance to cypermethrin compared with the susceptible SUD2S strain. The resistance to cypermethrin in the selected Nanjing strain increased to 2272 fold after several selections with cypermethrin. PASA genotyping results indicated that 100% individuals from the selected Nanjing strain had L 925I mutation (61.1% homozygotes and 38.9% heterozygotes), whereas only 75% individuals from the unselected Nanjing strain had L925I mutation (35% homozygotes, 40% heterozygotes and 25% wild type). Our results indicated that L925I mutation was tightly associated with pyrethroid resistance in the tobacco whitefly from Nanjing. Possible metabolic mechanisms for pyrethroid resistance in this strain were discussed.