<p><i>Brevipalpus yothersi</i> Baker is one of the main mite pests in commercial sweet orange orchards in Brazil and is the primary vector of citrus leprosis virus C (CiLV-C). Control of this mite has been predominantly based on synthetic acaricides. Recently, isocycloseram, a novel compound from the isoxazoline chemical group with both insecticidal and acaricidal activity, was registered in Brazil for the control of various pests, including <i>B. yothersi</i>. This study aimed to establish a baseline susceptibility of <i>B. yothersi</i> field populations to isocycloseram, providing critical information for the development of resistance monitoring and management strategies within integrated pest management (IPM) programs. A contact bioassay method was standardized for females using jack bean leaf discs. Nineteen field populations of <i>B. yothersi</i> collected from different citrus-growing regions in Brazil, along with a laboratory reference population, were evaluated. Median lethal concentrations (LC₅₀) ranged from 0.17 to 2.20 mg L^− 1, with resistance ratios (RR₅₀) between 0.22 and 2.78-fold, indicating low resistance levels. In contrast, LC₉₅ values ranged from 2.86 to 264.64 mg L^− 1, with RR₉₅ values from 7.51 to 92.53-fold, revealing resistance variation from low to very high among populations. The results indicate that susceptibility to isocycloseram in <i>B. yothersi</i> populations varied naturally and biogeographically, and not necessarily due to prior field exposure to the compound. The findings from this study provide essential technical support for the establishment of resistance monitoring programs for isocycloseram, aiming to ensure the sustainable use of this acaricide for controlling the CiLV-C vector mite in Brazilian citrus orchards.</p>