Acaricide resistance monitoring in phytophagous mites is crucial for effective pest management. Citrus growers in Cukurova region, a region that comprised more than 75% of total citrus production in Turkey, complained that acaricides have become inefficient in controlling outbreaks of the citrus red mite, Panonychus citri McGregor (Acari: Tetranychidae). To assess whether such control failures were caused by acaricide resistance, we bio-assayed P. citri populations collected from nine commercial orchards and a susceptible laboratory strain. Based on the mode of action (MoA) of acaricides, protonymphs were bio-assayed with etoxazole, spirodiclofen and spirotetramat, whereas abamectin and pyridaben were used for adults. Additionally, we determined kinetic esterase and glutathione S-transferase (GST) enzyme activities using a micro plate reader. Based on LC50 values, the resistance ratios of the orchard populations compared to the laboratory population varied from 2.81 to 34.82-fold, 1.22–18.35-fold, 1.23–40.43-fold, 1.76–27.50-fold and 2.24–75.06-fold for abamectin, etoxazole, spirodiclofen, spirotetramat and pyridaben, respectively. Total esterase and GST activities of the populations were in the ranges of 15.86–43.28 and 2.07–5.29 mOD/min/mg protein, respectively. The highest enzyme activity levels were detected in the Yakapinar population which was among one of the most resistant populations to all tested acaricides. The activities of detoxification enzymes may play an important role in the metabolic breakdown of the acaricides in resistant P. citri populations. Rotation of acaricides with different modes of action is fundamental to prevent potential resistance problems.