The effect of plant phenology and canopy structure of four crops and four weed species on reflectance spectra were evaluated in 2016 and 2017 using in situ spectroscopy. Leaf-level and canopy-level reflectance were collected at multiple phenologic time points in each growing season. Reflectance values at 2 wk after planting (WAP) in both years indicated strong spectral differences between species across the visible (VIS; 350–700 nm), near-infrared (NIR; 701–1,300 nm), shortwave-infrared I (SWIR1; 1,301–1,900 nm), and shortwave-infrared II (SWIR2; 1,901–2,500 nm) regions. Results from this study indicate that plant spectral reflectance changes with plant phenology and is influenced by plant biophysical characteristics. Canopy-level differences were detected in both years across all dates except for 1 WAP in 2017. Species with similar canopy types (e.g., broadleaf prostrate, broadleaf erect, or grass/sedge) were more readily discriminated from species with different canopy types. Asynchronous phenology between species also resulted in spectral differences between species. SWIR1 and SWIR2 wavelengths are often not included in multispectral sensors but should be considered for species differentiation. Results from this research indicate that wavelengths in SWIR1 and SWIR2 in conjunction with VIS and NIR reflectance can provide differentiation across plant phenologies and, therefore should be considered for use in future sensor technologies for species differentiation.