We show that monodisperse iron oxide nanoparticles in the size range of 6–17 nm, coated with brush-like layers of polystyrene or polyisoprene of different molecular weights, form well-defined lyotropic liquid crystalline phases. The lyotropic phase behaviour was investigated by synchrotron small-angle X-ray scattering. With increasing concentration, the polymer-coated nanoparticles show a disorder–order transition into well ordered bcc- or fcc-phases, depending on nanoparticle size and polymer molecular weights. The lyotropic phases can be shear oriented to obtain macroscopic highly ordered single crystalline nanoparticle superlattices exhibiting more than 100 Bragg-peaks. Within the ordered phases, the distance between adjacent nanoparticles can be varied systematically via concentration and attached polymer molecular weights in a range of 10–40 nm. We further demonstrate the versatility of lithographically patterned microstructured sample holders in combination with microfocus X-ray beams, which allow the investigation of very small sample volumes.