A hand-held hyperspectral radiometer was used to measure differences in reflectance characteristics of 24 Palmer amaranth and 15 pitted morningglory accessions collected from the central and southern United States. A hyperspectral reflectance reading was collected from two mature leaves at 24 and 27 d after emergence (DAE) for each accession. Two analysis techniques, linear discriminant analysis and best spectral-band combination (BSBC) analysis, were performed using various vegetation indices, spectral bands, and individual wavelengths. Differentiation of individual accessions was difficult. Palmer amaranth accession classification accuracies were < 50% using both analysis techniques, except one accession collected in South Carolina (63%), when pooled over data acquisition dates. Pitted morningglory accession classification accuracies were also generally < 50%. Classification accuracies were higher using BSBC analysis at 24 DAE; however, at 27 DAE only one accession resulted in classification accuracy > 30%. These results suggest there are only slight reflectance differences within Palmer amaranth and pitted morningglory accessions. These differences may not be predictable based upon accession origin because of the genetic diversity of Palmer amaranth and pitted morningglory. However, differentiation between Palmer amaranth and pitted morningglory was 100%. Thus, spectral sensors used to differentiate between Palmer amaranth and pitted morningglory do not need to be calibrated for a particular region of the United States, and differentiation between these two species can be made using reflectance characteristics.