Analyzing the chemical space coverage in commercial fragment screening collections revealed the overlap between bioactive medicinal chemistry substructures and rule-of-three compliant fragments is only ∼25%. We recommend including these fragments in fragment screening libraries to maximize confidence in discovering hit matter within known bioactive chemical space, while incorporation of nonoverlapping substructures could offer novel hits in screening libraries. Using principal component analysis, polar and three-dimensional substructures display a higher-than-average enrichment of bioactive compounds, indicating increasing representation of these substructures may be beneficial in fragment screening.