The two-spotted spider mite Tetranychus urticae Koch (TSSM), a cosmopolitan and profoundly polyphagous pest with over 1200 plant hosts, is known to be prone to frequently developing acaricide resistance. Traits, such as rapid development, short generation time and high fecundity provide favorable conditions for acquiring resistance and have placed this species at the forefront of reported pesticide resistance cases. TSSM resistance presents a global issue, notably in greenhouse plant production. This species is a ubiquitous agricultural pest present in the acarofauna of Serbia. TSSM resistance has been observed and characterized worldwide, but has not been studied in Serbia to date. The main objective of this pilot study was to survey the presence of several known acaricide resistance-conferring target-site mutations and assess their contribution to reported ineffective treatment of TSSM populations in commercial greenhouses of Serbia. This survey is an initial endeavor which will serve as a starting point for a more comprehensive study of pesticide resistance of TSSM populations in Serbia. Five geographically distinct TSSM greenhouse populations from various plant hosts were selected for this study. All populations had previously been unsuccessfully treated with abamectin, two populations additionally with etoxazole and hexythiazox, respectively. Samples were collected in infestation hotspots. Mites were manually selected and preserved in 96% ethanol. Genomic DNA was extracted from 100 pooled and crushed adult mites with E.Z.N.A. Tissue DNA Kit. Species identification was achieved via BOLD Identification Engine analysis of COI and ITS2 barcode sequences. Screening of target-site mutations was conducted on regions of CHS1, GluCl1 and GluCl3. All sequences were obtained via PCR amplification, commercial purification and Sanger sequencing. Sequencing chromatograms were visually inspected in BioEdit v.7.7.1 and aligned with reference sequences using ClustalW in MEGA 12. Mutations were characterized as detected or not detected. A known resistance-conferring amino acid substitution of CHS1, I1017F, was detected in the only examined population reported as unsuccessfully treated with hexythiazox. Additionally, an I321T mutation of GluCl3 was detected in one of the populations. Results indicate that known acaricide target-site mutations may have contributed to ineffective treatment and resistance development in two of the examined populations. Further research on an expanded dataset, additionally including toxicity and enzyme assays, needs to be conducted for a more complete overview.