Ligand-gated ion-channel N-methyl-D-aspartate receptors (NMDARs) are major excitatory receptors in the brain and have critical developmental and maintenance roles in the central nervous system. Most studies of recombinant NMDARs have focused on diheteromeric receptors that contain two copies of GluN1 and two identical GluN2 subunits. However, diverse GluN2-subunits are expressed in most neurons, and there is strong evidence suggesting many native NMDARs are triheteromeric assemblies of two GluN1 and two different GluN2 subunits (e.g. GluN1/GluN2A/GluN2D or any other combination). Receptors that contain two different subunits, such as GluN2A and GluN2D, may have unique pharmacological and functional properties that are distinct from the corresponding diheteromeric assemblies. Elucidating triheteromeric NMDAR pharmacology has been impeded by limited methods to express triheteromeric assemblies without diheteromeric receptors in a heterologous system. We have adapted a strategy using the masking of the endoplasmic reticulum retention signal to selectively express triheteromeric GluN1/GluN2A/GluN2D receptors with minimal contribution of diheteromeric assemblies (“escape currents”) in Xenopus laevis oocytes. We have profiled expression of GluN1/GluN2A/GluN2D receptors and their “escape current” controls over a 4-5-day period post-injection and found that optimal triheteromeric expression happens on the 4th day post-injection for this triheteromeric combination. Triheteromeric GluN1/GluN2A/GluN2D receptors have distinct pharmacological profile in experiments with selective and non-selective agonists, endogenous ions (zinc, magnesium), and allosteric modulators, demonstrated by distinct EC50/IC50 values compared to the diheteromeric GluN1/GluN2A and GluN1/GluN2D receptors. For example, GluN1/GluN2A/GluN2D receptors had intermediate EC50 values for glutamate and glycine. Interestingly, as seen for other triheteromeric NMDARs, a single copy of GluN2A was sufficient to exhibit zinc-mediated inhibition in GluN1/GluN2A/GluN2D NMDARs. This study will advance our understanding of triheteromeric NMDAR function and pharmacology, and aid in development of compounds with therapeutic potential.