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BACKGROUNDThe acaricides cyflumetofen, cyenopyrafen and pyflubumide inhibit the mitochondrial electron transport chain at complex II [succinate dehydrogenase (SDH) complex]. A target site mutation H258Y was recently discovered in a resistant strain of the spider mite pest Tetranychus urticae. H258Y causes strong cross‐resistance between cyenopyrafen and pyflubumide, but not cyflumetofen. In fungal pests, fitness costs associated with substitutions at the corresponding H258 position that confer resistance to fungicidal SDH inhibitors have not been uncovered. Here, we used H258 and Y258 near‐isogenic lines of T. urticae to quantify potential pleiotropic fitness effects on mite physiology.RESULTSThe H258Y mutation was not associated with consistent significant changes of single generation life history traits and fertility life table parameters. In contrast, proportional Sanger sequencing and droplet digital polymerase chain reaction showed that the frequency of the resistant Y258 allele decreased when replicated 50:50 Y258:H258 experimentally evolving populations were maintained in an acaricide‐free environment for approximately 12 generations. Using in vitro assays with mitochondrial extracts from resistant (Y258) and susceptible (H258) lines, we identified a significantly reduced SDH activity (48% lower activity) and a slightly enhanced combined complex I and III activity (18% higher activity) in the Y258 lines.CONCLUSIONOur findings suggest that the H258Y mutation is associated with a high fitness cost in the spider mite T. urticae. Importantly, while it is the most common approach, it is clear that only comparing life history traits and life table fecundity does not allow to reliably estimate fitness costs of target site mutations in natural pest populations. © 2023 Society of Chemical Industry.