It is challenging to incorporate stereochemical diversity and topographic complexity into DNA-encoded libraries (DELs) because DEL syntheses cannot fully exploit the capabilities of modern synthetic organic chemistry. Here, we describe the design, construction, and validation of DOS-DEL-1, a library of 107 616 DNA-barcoded chiral 2,3-disubsituted azetidines and pyrrolidines. We used stereospecific C–H arylation chemistry to furnish complex scaffolds primed for DEL synthesis, and we developed an improved on-DNA Suzuki reaction to maximize library quality. We then studied both the structural diversity of the library and the physicochemical properties of individual compounds using Tanimoto multifusion similarity analysis, among other techniques. These analyses revealed not only that most DOS-DEL-1 members have “drug-like” properties, but also that the library more closely resembles compound collections derived from diversity synthesis than those from other sources (e.g., commercial vendors). Finally, we performed validation screens against horseradish peroxidase and carbonic anhydrase IX, and we developed a novel, Poisson-based statistical framework to analyze the results. A set of assay positives were successfully translated into potent carbonic anhydrase inhibitors (IC50 = 20.1–68.7 nM), which confirmed the success of the synthesis and screening procedures. These results establish a strategy to synthesize DELs with scaffold-based stereochemical diversity and complexity that does not require the development of novel DNA-compatible chemistry.