Sitophilus zeamais (Motschulsky) (Coleoptera: Curculionidae) is one of the economically important insect pests that infest stored products. In order to control this pest, chemical insecticides, such as malathion, have been widely used as grain protectants in many countries including Turkey. However, continuous application of insecticides at high doses has resulted in the development of resistance in this pest. Hence, this study aims to determine the toxicity of malathion insecticide at discriminating dose on S. zeamais populations and to analyse the role of detoxification enzyme systems in the development of resistance in S. zeamais populations in Turkey. Bioassay results showed that while S. zeamais Ankara population showed resistance to malathion insecticide, S. zeamais Samsun population did not show resistance to the insecticide. In addition, biochemical analysis indicated that CYP450-PNOD activities showed 2.0-fold increase in malathion resistant Ankara population. Although, EST-PNPA (1.3-fold) and GST-DCNB (1.5-fold) activities significantly increased in S. zeamais Samsun population, they did not show any significant changes in malathion resistant S. zeamais Ankara population. Consequently, it seems probable that cytochrome P450 monooxygenases play an active role in malathion resistance in Ankara population of S. zeamais.