The translocator protein 18 kDa (TSPO) remains a cornerstone biomarker for imaging neuroinflammation and microglial activation via positron emission tomography (PET), but widespread clinical translation has been hindered by binding sensitivity to the common human TSPO rs6971 (A147T) polymorphism. All clinically used TSPO PET ligands exhibit significant affinity loss for the A147T isoform, necessitating genotype stratification in clinical studies. To address this limitation at the molecular level, we undertook a focused medicinal chemistry campaign to design, synthesize, and evaluate novel pyrazolo[1,5-a]pyrimidine-based TSPO ligands with altered acetamide substituents to enhance binding across genotypes. Guided by structure–activity relationships and CNS multiparameter optimization principles, we identified DPA-813 and its fluorinated analogue DPA-814, both demonstrating single-digit nanomolar affinity for wild-type and A147T TSPO in vitro, and substantially improved binding versus parent compounds. These scaffolds were successfully radiolabelled as [11C]DPA-813 and [18F]DPA-814 with high purity and molar activity, showed robust specific binding across human brain tissues irrespective of rs6971 status, favorable stability, and brain penetration in rodents, and displayed promising neuroinflammation imaging in EAE models. This talk will dissect the key medicinal chemistry strategies that enabled polymorphism insensitivity and outline next steps we have taken toward first-in-human PET evaluation for [18F]DPA-814.