PCR-RFLP assays were developed for detecting the L925I and the F331W resistance mutations. A highly specific PASA was developed for detecting the T929V mutation. The molecular diagnostic tools were used to monitor the frequency of the resistance mutations in a large number of field caught Q biotype B. tabaci from Crete (Greece), where both organophosphates and pyrethroids are extensively used. The F331W mutation was fixed in all field individuals examined. The pyrethroid resistance mutations were detected in high frequencies: 0.38 and 0.54 for L925I and T929V, respectively. The simple diagnostics are accurate and robust, to be used alongside classical bioassays to prevent ineffective insecticide applications, and for early identification of the spreading of resistant Q biotype populations into new regions around the globe.