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ABSTRACT High‐grading bias is the overestimation power in a subset of loci caused by model overfitting. Using both empirical and simulated datasets, we show that high‐grading bias can cause severe overestimation of population structure, and thus mislead investigators, whenever highly informative or high‐F_STmarkers are chosen (i.e., ascertained) and used for subsequent assessments, a common practice in population genetic studies. This problem can occur in panmictic populations with no local adaptation.Biased results from choosing high‐F_STmarkers may have severe downstream implications for management and conservation, such as erroneous conservation unit delineation, which could squander limited conservation resources to protect incorrectly defined ‘populations’. Furthermore, we caution that high‐grading is not limited toF_STapproaches; high‐grading bias is a concern whenever a small subset of markers are first chosen to explain differences among groups based on their degree of difference and are subsequently reused to estimate the degree of difference among those groups. For example, selecting highF_STloci for use in a GT‐seq panel or using differentially expressed genes to plot sample membership in multivariate space can both result in spurious structure when none exists. We illustrate that using statistically based outlier tests in place of arbitraryF_STcut‐offs can reduce bias. Alternatively, permutation tests or cross‐evaluation can be used to detect high‐grading bias. We provide an R package, PCAssess, to help researchers detect and prevent high‐grading bias in genetic datasets by automating permutation tests and principal component analyses (https://github.com/hemstrow/PCAssess).