The ability to differentiate a non-native aquatic plant, Myriophyllum spicatum (Eurasian watermilfoil or EWM), from other submerged aquatic vegetation (SAV) using spectral data collected at multiple scales was investigated as a precursor to mapping of EWM. Spectral data were collected using spectroradiometers for SAV taken out of the water, from the side of a boat directly over areas of SAV and from a lightweight portable radiometer system flown from an unmanned aerial system (UAS). EWM was spectrally different from other SAV when using 651 spectral bands collected in ultraviolet to near-infrared range of 350 to 1000 nm but does not provide a practical system for EWM mapping because this exceeds the capabilities of available airborne hyperspectral imaging systems. Using only six spectral bands corresponding to an available multispectral camera or eight wetlands-centric bands did not reliably differentiate EWM from other SAV and assemblages. However, a modified version of the normalized difference vegetation index (mNDVI), using a ratio of red-edge to red light, was significantly different among dominant vegetation groups. Also, averaging the full range of spectral to 65 10-nm wide bands, similar to available hyperspectral imaging systems, provided the ability to identify EWM separately from other SAV. The UAS-collected spectral data had the lowest remote sensing reflectance versus the out-of-water and boatside data, emphasizing the need to collect optimized data. The spectral data collected for this study support that with relatively clear and calm water, hyperspectral data, and mNDVI, it is likely that UAS-based imaging can help with mapping and monitoring of EWM.