The two-spotted spider mite, Tetranychus urticae (Koch, 1836) (Acari: Tetranychidae) causes significant problems in cotton fields. The pest has developed resistance to abamectin, which is used intensely against spider mite populations. A discriminating dose was applied to T. urticae populations collected from Aydın, Adana, Şanlıurfa, and Diyarbakır provinces between 2019 and 2021, where there is significant cotton production and intensive acaricide applications. Populations with a mortality rate of less than 80% at the discriminating dose applied to field populations were included in the bioassay study. Then, the 10 most resistant populations were determined based on their LC50 and LC90 values. It was observed that there was high resistance (HR) in two T. urticae populations collected from Diyarbakır (DIY28 and DIY2) with LC50 values of 24.5 and 33.81 mg L-1, respectively 113 and 156-fold and one population collected from the province of Aydın (AYD4) LC50 value of 23.5 mg L-1 and 109-fold. As a result of the comparisons with a susceptible population in biochemical studies, it was determined that the carboxylesterase (CarE) enzyme of these populations (except for SAN6, SAN7, SAN8, ADA9, and ADA11) had more than 2-fold higher activity compared to the glutathione enzyme (GST), and they were in statistically different groups. When the GST enzyme activities were examined, it was found that the enzyme activities were generally low, except for the ADA11 and ADA16 populations (3.73 and 2.3-fold). In terms of the target site resistance, it was observed that there was no target site mutation (G314D and I321T-G326E) in the glutamate-gated chloride channels (Units GluCl 1 and 3) in these highest 10 resistant populations. It can be said that the resistance levels determined for abamectin from the obtained populations were determined to be low. In brief, out of the total 44 populations brought in and tested, only 30 populations were considered resistant with a mortality rate below 80% after the applied discriminating dose. It was determined that 6.8% of these 30 populations had HR, 31.8% had medium resistance (MR), and 29.5% had low resistance (LR). When examining by regions, it was found that high resistance populations were more prevalent in Diyarbakır and Aydın (15.4% and 10%, respectively) provinces, while the highest percentage of MR populations was found in Şanlıurfa, at 90%. Abamectin still acts as an effective acaricide. However, it is recommended to use acaricides with different modes of action to prevent possible emergence of resistance cases.