This study investigated the population structure and genetic diversity of the two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), across three different environments: farm, greenhouse and laboratory. Analysis of a 632-nucleotide fragment of the mitochondrial COI gene revealed four distinct haplotypes, with one haplotype shared among all populations, suggesting a common genetic origin. The farm population exhibited the highest haplotype diversity (Hd = 0.699), while the greenhouse population showed minimal diversity and the laboratory population exhibited complete uniformity. Genetic distance analysis confirmed these differences, showing significant divergence between the farm population and the more genetically uniform greenhouse and laboratory groups. ISSR marker analysis supported these findings, with the farm population having the highest number of alleles (4) and effective alleles, as well as elevated values for Shannon's information index and expected heterozygosity. Principal Coordinates Analysis (PCoA) revealed clear genetic clustering: laboratory samples formed a tight isolated cluster, greenhouse samples showed moderate clustering and farm samples were broadly dispersed. These patterns indicate that rearing environments significantly influence the genetic structure of T. urticae, with open field conditions promoting genetic diversity, while controlled environments lead to reduced variation. These findings are important for understanding pest adaptability and optimizing integrated pest management (IPM) strategies.