The European red mite, Panonychus ulmi Koch, is one of the major pests of apple trees in Iran as well as in the world. By feeding on apple parenchymal cells, P. ulmi can significantly reduce photosynthesis and disrupt general plant activity which often leads to a significant reduction in the quality and quantity of apple fruits. Regarding the high damage ability of the mite, its chemical control is often unavoidable to prevent crop loss. Spirodiclofen, a specific and non-systemic acaricide, belongs to tetronic acids and acts by inhibiting acetyl coenzyme-A-carboxylase. One of the importanat problems in the pest chemical control is the resistance development. Resistance is a heritable ability in an insect or mite strain to tolerate lethal doses of a pesticide. In order to evaluate P. ulmi resistance to spirodiclofen, several populations of P. ulmi were collected from apple orchards of Iran including: West Azarbaijan (Urmia, Shahin Dezh, Mahabad, Salmas), East Azarbaijan (Miyane, Marand, Maragheh, Ahar) and Isfahan (Semirom). The average lethal doses (LC50s) of different populations were measured by bioassay tests using a Potter spray tower. The highest resistance to spirodiclofen observed in Urmia and Shahin dezh populations with the LC50 values of 433.26 (mg a.i/L) and 305.88 (mg a.i/L) and with resistance ratios of 214.5 and 149.4, respectively. Ahar population was the most susceptible with an LC50 value of 2.1 (mg a.i./L). To elucidate the resistance mechanisms, the role of detoxyfing enzymes, Cytochrome P450 monoxygenases, esterases and glutathione-S-transferases, were determined using the synergist. Therefore, the effects of pretreatment with three synergists, pipronil Botoxide (PBO), triphenilphosphat (TPP) and diethyl maleat (DEM), were evaluated on the resistant and susceptible populations. PBO pretreatment caused higher reduction in the LC50 values than that of other synergists. In addition, PBO reduced the resistance ratios. Inhibiting esterases and glutathione-S-transferases with TPP and DEM, respectively, showed that these enzymes have no major role in the spirodiclofen resistance. In conclusion, possibly enhanced detoxification, especially P450s play an important role in spirodiclofen resistance in P. ulmi. The results can be used in the early detection of spirodiclofen resistance in P. ulmi populations and preventing or delaying the resistance development and finally it may help in reducing acaricide applications and environment damages.