Anopheles sacharovi and Anopheles superpictus have a significant public health importance since they are primer and seconder malaria vectors of Turkey, respectively. As a result of intensive insecticide usage in historically malaria endemic regions of Turkey for long years, insecticide resistance problem has occurred inevitably. In this study, we aimed to investigate the involvement of the detoxification enzymes in insecticide resistance in Turkish An. sacharovi and An. superpictus populations in the Mediterranean and South-eastern Anatolia region where have a malaria history in the past. Bioassay results indicated that both An. sacharovi and An. superpictus populations are resistant to DDT, resistant or possible resistant to organophosphates and carbamates and finally mostly susceptible to pyrethroids. Although bioassays results indicated high DDT resistance in all mosquito populations, biochemical assays did not show significantly high GST levels in all strains. Almost all An. sacharovi and An. superpictus populations had an increased α and β esterase activity levels while nearly half of the overall populations had an increased p-NPA esterase than the control group. Elevated levels of MFO frequency have been shown in the majority of the populations. Consequently, our results reveal that biochemical resistance mechanisms may play an important role in insecticide resistance in Turkish An. sacharovi and An. superpictus populations. These results give useful cues to monitor the insecticide resistance before it spreads throughout an entire population, enabling early intervention.