N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors found ubiquitously in the nervous system. Mutations in genes encoding NMDARs are associated with a broad range of neurological and psychiatric disorders, but their causes at the protein level remain elusive. To study the structural changes in NMDARs caused by disease-associated mutations we employed single-molecule FRET, a technique that allows us to observe conformational changes in real-time by measuring precise distances between loci labelled with a pair of fluorophores, one of which emits light proportionate to their proximity. We studied labelled wild-type and mutant rat NMDARs expressed in HEK293T cells, harvested, and isolated on a coverslip; and recorded the FRET signal using TIRF microscopy. We demonstrated that the conformational dynamics at the beginning of the amino-terminal domain upon application of ligands previously reported in the literature are not affected by mutations that have uncertain clinical significance but are dramatically disrupted in receptors carrying the I150V mutation in the GluN2B subunit, which is widely reported as pathogenic. Furthermore, we identified two loci deeper within the protein structure suitable for smFRET labelling and demonstrated that the conformational dynamics of receptors with the I150V mutation are disrupted considerably at one of those sites (E342, GluN1) but only marginally at the other (I691, GluN1). Our findings show that disease-associated mutations can have a measurable and localised effect on the structural dynamics of NMDARs, which in the future could be combined with studies of functional outcomes (e.g. electrophysiology) to elucidate the exact mechanism of neurological disorders.