The precise spatial coordination of protein complexes is essential for cellular function, particularly in neuronal synapses where proteins responsible for neurotransmission form subsynaptic regions of high density called nanoclusters. These clusters, whose trans- cellular alignment modulates synaptic signal propagation, include NMDARs which are crucial for learning and memory. However, the spatial relationships between these proteins and NMDARs, as well as the specific configurations of NMDAR subunits within these clusters, remain poorly defined. Using multiplexed super-resolution DNA-PAINT microscopy, We mapped the relationship of key NMDAR subunits, GluN2A and GluN2B, to reference proteins in the active zone and postsynaptic density. Our findings reveal that GluN2A and GluN2B subunits form nanoclusters with diverse configurations that, on average, are not localized near presynaptic vesicle release sites marked by Munc13-1. Notably, a subset of presynaptic sites is structured to maintain NMDAR activation, characterized by denser interiors, alignment with abundant PSD-95, and close association with specific NMDAR nanodomains. Additionally, NMDARs, which are heterotetramers composed of GluN1, GluN2A-D, or GluN3A-B subunits, exhibit functional diversity due to their subunit composition. Triheteromeric NMDARs, containing both GluN2A and GluN2B subunits, display unique channel kinetics and agonist sensitivity compared to diheteromeric receptors. To accurately identify and study these triheteromeric NMDARs, we developed a novel probe using bimolecular complementation by attaching split-reporters to the N-terminal domains of GluN2A and GluN2B. This method produces fluorescence exclusively in the presence of triheteromeric NMDARs, ensuring specificity. Our results demonstrate that split-taggedNMDARs preserve normal receptor function and efficiently traffic to synapses, particularly those enriched with PSD-95. This innovative tool sheds light on the subcellular distribution and synaptic incorporation of triheteromeric NMDARs, advancing our understanding of their role in synaptic transmission and neurological disorders. Our combined findings reveal a new principle regulating NMDAR signaling, highlighting the significance of multiprotein nanodomains whose internal architecture is contingent on trans-cellular interactions. This research enhances our understanding of synaptic functional architecture and the intricate mechanisms underlying synaptic transmission and plasticity.