This study aims to determine the resistance ratios of Tetranychus urticae Koch red form (RF) and Tetranychus turkestani (Ugarov & Nikolskii) populations collected from cotton fields in Soke, Germencik, Kocarli and Nazilli districts of Aydin province to commonly used acaricides (abamectin, hexythiazox, etoxazole, spiromesifen). The potential role of detoxification enzymes (esterase, glutathione-s-transferase (GST), P450 monooxygenase (P450)) in acaricide resistance was also assessed. Overall populations of T. urticae RF presented varying ratios of resistance to these acaricides with all three populations (G15R21, K24R21, S3R21) showing significant resistance to abamectin (187.1–223.66 fold), hexythiazox (156.31–168.25 fold), etoxazole (409.58–517.20 fold) and spiromesifen (981.77–1246.11 fold). In contrast, all five T. turkestani populations (G4G21, K16G21, N1G21, N11G21, S19G21) were susceptible to abamectin, etoxazole, and spiromesifen, but exhibited some resistance to hexythiazox (164.34–182.39 fold). The resistance ratios of T. turkestani were however lower compared to T. urticae RF. A relationship was also found between detoxification enzymes and resistance. Esterase activity varied among mite populations, with the highest levels recorded in resistant populations of T. urticae (G15R21 and K24R219) with rates ranging between 6.48 and 7 mOD/min/mg protein. GST activity was highest in resistant mite populations (T. urticae S3R21 and T. turkestani K16G21). T. turkestani N11G21 population exhibited the highest P450 enzyme activity, reaching 4.39 RFU/30 min/mg protein. This study was the first to determine the resistance ratios of T. turkestani to hexythiazox in Türkiye, and on a global scale.