Ontario is Canada’s largest soybean producer, and its greenhouse sector accounts for 71% of national greenhouse production. Spider mites (Acari: Tetranychidae) are major agricultural pests due to their broad host range, rapid evolution of pesticide resistance, and capacity to cause severe crop damage. Management programs have historically focused on the two-spotted spider mite (TSSM), Tetranychus urticae (Koch), and its chemical control. However, there are knowledge gaps in mite diversity and resistance status, challenging current IPM strategies. Here, we provide an updated assessment of pesticide resistance levels and species composition in spider mite populations collected from greenhouses and soybean fields across Southern Ontario. The study analyzed 163 spider mite populations collected between 2018 and 2025 from greenhouses (86 samples from cucumber, eggplant, pepper, strawberry, and tomato) and soybean fields (77) across Southern Ontario, Canada. Pesticide susceptibility was assessed using bioassays tailored to each mode of action: a high-throughput soaking assay (adulticidal) for abamectin, bifenazate, and cyflumetofen; a leaf-disk dipping assay (ovicidal) for etoxazole, spiromesifen, and pyridaben; and a leaf-disk spray assay (adulticidal) for dimethoate. All bioassays were conducted at LC90 concentrations established for a susceptible reference population (LND), with mortality rates =40% considered indicative of resistance. Species identification was performed using morphological traits and molecular markers (ITS and COI). Of the 77 soybean populations screened, 49% were resistant to dimethoate. As dimethoate is the only registered product for TSSM control in Ontario soybeans, this widespread resistance is particularly concerning. Among 32 soybean populations further tested with additional pesticides, resistance to abamectin (3/32) and spiromesifen (4/32) was detected. Out of 46 greenhouse populations evaluated against seven pesticides, only 14 were fully susceptible to all compounds, and 45% exhibited multi-resistance. Across all populations tested, pyridaben was the most effective compound. In samples received, species identification revealed additional pest species, including a novel Tetranychus sp. nov. and Tetranychus turkestani Ugarov & Nikolskii, previously unreported in Ontario. The coexistence of multiple spider mite species with distinct feeding damage and resistance profiles increases the risk of control failures. With climate change likely to favour further spread and outbreak intensity, improved molecular diagnostics and species-specific IPM strategies will be essential for sustainable crop protection.