Abstract Class II major histocompatibility complex molecules (MHCII) are transmembrane glycoproteins that display peptides generated and selected by intracellular antigen processing and presentation mechanisms to CD4+T cells, initiating an adaptive immune response. MHCII is expressed on the cell surface and in endosomal compartments, each of which have their own signature membrane characteristics. The traditional strategy to investigate peptide binding to MHCII has relied on the use of soluble MHCII, in which the transmembrane portion has been removed. This work studied MHCII in its native, membrane-embedded form. To this end, we used nanodisc, a synthetic model membrane device, to evaluate the effect of membrane lipid composition on MHCII assembly. The full-length human MHCII allele HLA-DR1 (DR1) was isolated from B-lymphoblastoid cell lines via immunoaffinity chromatography. Three types of nanodisc were generated: simple, fluid disordered and rigid ordered, each of them characterized by a unique lipid composition. Nanodiscs were separated using fast protein liquid chromatography (FPLC), the readings from which showed clear differences between nanodisc types. We observed that the fluid disordered nanodisc showed DR1 assembly as a cluster, with one major FPLC-generated peak. In contrast, simple and rigid nanodiscs were distinguished by multiple peaks. As DR1 tends to form aggregates, we inferred that in simple and rigid nanodiscs DR1 tetramers and dimers form in separate nanodisc assemblies. Taken together, our results indicate that membrane lipid composition has a strong impact on native MHCII assembly. As such, we predict that MHCII conformation and activity are function of the cell compartment they reside at any point in time.