We have developed a protocol to isolate synaptic and extrasynaptic membranes from post-mortem human cortex. This allowed us to characterize the distribution of the major brain NMDAR subunits, GluN2B, GluN2A, GluN1 and GluN3A in controls and AD samples. We used specific antibodies and western blots to analyse the level and phosphorylation of the NMDAR subunits. We have found that GluN2B, GluN2A, GluN1 are predominantly at synaptic membranes, while GluN3A is more abundant at extrasynaptic membranes. When the synaptic distribution was analysed in AD fractions, GluN2B and GluN2A showed lower levels respect to controls. Remarkably, GluN2B and GluN1 levels at extrasynaptic membranes were significantly higher in AD fractions than in controls. GluN3A levels were lower in extrasynatic fractions from Braak stages V-VI only. We also identified glycoforms of GluN2B and GluN2A enriched at extrasynaptic membranes, being more abundant and harbouring a different glycosylation in AD fractions. GluN2B was phosphorylated at Tyr1336 in synaptic and extrasynaptic membranes, while phosphorylation at Tyr1472 was found almost exclusively at synaptic membranes, in controls and AD fractions. Interestingly, GluN2B phosphorylation at Tyr1472 was significantly low in AD synaptic membranes. In conclusion, we observed that NMDAR subunit distribution is affected in AD cases, particularly for GluN2B. The significant reduction of NMDAR subunit levels in AD synaptic membranes is likely contributing to the synaptic transmission failure that occurs in this pathology; additionally, the increment in specific NMDAR subunits at extrasynaptic membranes could be favouring the activation of excitotoxicity in AD.