Recently, mixture formulations of different active ingredients have been used to prevent or delay resistance in pests. However, their effect on resistance development has not been extensively studied in spider mites. Therefore, the development of resistance to a spiromesifen+abamectin (SA) mixture in Panonychus ulmi populations from apple orchards in Isparta, Türkiye, and a laboratory-selected population was investigated in this study. Cross-resistance to spiromesifen, spirodiclofen, abamectin, milbemectin, and spirodiclofen+abamectin was also evaluated. Three orchard populations showed low resistance (3.2–7.6-fold), while the laboratory-selected population (IR) exhibited moderate resistance (23.1-fold) to the SA mixture. Synergist assays with DEM, TPP, and PBO revealed significant involvement of detoxification enzymes (esterases, GST, and P450). Molecular analyses identified G314D (GluCl1) and I321T (GluCl3) previously characterized point mutations associated with abamectin resistance, and several novel mutations in acetyl-CoA carboxylase (ACCase) domains potentially linked to spiromesifen resistance. These results indicate that both metabolic and target-site mutation mechanisms contribute to the resistance determined in P. ulmi.