The frequent application of acaricides is necessary for the management of the two-spotted spider mite Tetranychus urticae because of its high tendency to rapidly develop resistance to the used chemicals. Here, we aimed to investigate the underlying mechanisms of the simultaneous development of acaricide resistance in multiple mite populations and assessing their genetic structure. To this end, we used four microsatellite loci to analyze the genetic structure of 11 T. urticae populations collected from eight commercial apple orchards, one apple tree nursery, one experimental apple orchard, and one hop farm. In the commercial orchards and nursery, acaricides were used frequently every year; in the experimental apple orchard, acaricides were not used after transplantation; and in the hop farm, a propylene glycol emulsion was used periodically. Allelic richness and heterozygosity were not highly different among the orchards. In most mite populations, the inbreeding coefficients were significantly higher than zero, indicating frequent occurrence of inbreeding. Inbreeding coefficient values were higher in populations collected from commercial orchards, suggesting that acaricide applications increased the frequency of inbreeding in mite populations. Overall, the results showed that the recurrent chemical treatments have a strong bottleneck effect on T. urticae populations, which probably accelerates the development of acaricide resistance.