The foundation of contemporary weed management practices in many parts of the world is glyphosate. However, dependency on the effectiveness of herbicide practices has led to overuse through continuous growth of crops resistant to a single mode of action. In order to provide a cost-effective weed management strategy that does not promote glyphosate-resistant weed biotypes, differences between resistant and susceptible biotypes have to be identified accurately in the field conditions. Unmanned Aerial Vehicle (UAV)-assisted thermal and multispectral remote sensing has potential for detecting biophysical characteristics of weed biotypes during the growing season, which includes distinguishing glyphosate-susceptible and glyphosate-resistant weed populations based on canopy temperature and deep learning driven weed identification algorithms. The objective of this study was to identify herbicide resistance after glyphosate application in true field conditions by analyzing the UAV-acquired thermal and multispectral response of kochia, waterhemp, redroot pigweed, and common ragweed. The data were processed in ArcGIS for raster classification as well as spectral comparison of glyphosate-resistant and glyphosate-susceptible weeds. The classification accuracy between the sensors and classification methods of maximum likelihood, random trees, and Support Vector Machine (SVM) were compared. The random trees classifier performed the best at 4 days after application (DAA) for kochia with 62.9% accuracy. The maximum likelihood classifier provided the highest performing result out of all classification methods with an accuracy of 75.2%. A commendable classification was made at 8 DAA where the random trees classifier attained an accuracy of 87.2%. However, thermal reflectance measurements as a predictor for glyphosate resistance within weed populations in field condition was unreliable due to its susceptibility to environmental conditions. Normalized Difference Vegetation Index (NDVI) and a composite reflectance of 842 nm, 705 nm, and 740 nm wavelength managed to provide better classification results than thermal in most cases.