The subthalamic nucleus (STN) is crucial for normal motor, limbic and associative function. STN dysregulation is correlated with several brain disorders, including Parkinsons disease and obsessive compulsive disorder (OCD), for which high-frequency stimulation of the STN is increasing as therapy. However, clinical progress is hampered by poor knowledge of the anatomical-functional organization of the STN. Today, experimental mouse genetics provides outstanding capacity for functional decoding, provided selective promoters are available. Here, we implemented single-nuclei RNA sequencing (snRNASeq) of the mouse STN followed through with histological analysis of 16 candidate genes of interest. Our results demonstrate that the mouse STN is composed of at least four spatio-molecularly defined domains, each distinguished by defined sets of promoter activities. Further, molecular profiles dissociate the STN from the adjoining para-STN (PSTN) and neighboring structures of the hypothalamus, mammillary nuclei and zona incerta. Enhanced knowledge of STNs internal organization should prove useful towards genetics-based functional decoding of this clinically relevant brain structure. p id=Par Wallen-Mackenzie et al. investigate anatomical-functional organization of the subthalamic nucleus in mice, using single-nuclei RNA sequencing followed by histological analysis. They identify four domains distinguished by defined sets of promoter activities, providing a valuable resource for functional decoding of the subthalamic nucleus.