The application of hyperspectral imaging technology for plant disease detection in the field is still challenging. Existing equipment and analysis algorithms are adapted to highly controlled environmental conditions in the laboratory. However, only real time information from the field scale is able to guide plant protection measures and to optimize the use of resources. At the field scale, many parameters such as the optimal measurement distance, informative feature sets, and suitable algorithms have not been investigated. In this study, the hyperspectral detection and quantification of yellow rust in wheat was evaluated using two measurement platforms: a ground-based vehicle and an unmanned aerial vehicle (UAV). Different disease development stages and disease severities were provided in a plot-based field experiment. Measurements were performed weekly during the vegetation period. Data analysis was performed by three prediction algorithms with a focus on the selection of optimal feature sets. In this context, the across-scale application of optimized feature sets, an approach of information transfer between scales, was also evaluated. Relevant aspects for an on-line disease assessment in the field integrating affordable sensor technology, sensor spatial resolution, compact analysis models, and fast evaluation have been outlined and reflected upon. For the first time, a hyperspectral imaging observation experiment of a plant disease was comparatively performed at two scales, ground canopy and UAV.