Giant ragweed is a highly competitive weed that continually threatens crop production systems due to evolved resistance to acetolactate synthase–inhibiting herbicides (ALS-R) and glyphosate (GR). Two biotypes of GR giant ragweed exist and are differentiated by their response to glyphosate, termed here as rapid response (RR) and non–rapid response (NRR). A comparison of data from surveys of Indiana crop fields done in 2006 and 2014 showed that GR giant ragweed has spread from 15% to 39% of Indiana counties and the NRR biotype is the most prevalent. A TaqMan® single-nucleotide polymorphism genotyping assay was developed to identify ALS-R populations and revealed 47% of GR populations to be ALS-R as well. The magnitude of glyphosate resistance for NRR populations was 4.6 and 5.9 based on GR50 and LD50 estimates, respectively. For RR populations, these values were 7.8 to 9.2 for GR50 estimates and 19.3 to 22.3 for LD50 estimates. A novel use of the Imaging-PAM fluorometer was developed to discriminate RR plants by assessing photosystem II quantum yield across the entire leaf surface. H2O2 generation in leaves of glyphosate-treated plants was also measured by 3,3′-diaminobenzidine staining and quantified using imagery analysis software. Results show photo-oxidative stress of mature leaves is far greater and occurs more rapidly following glyphosate treatment in RR plants compared with NRR and glyphosate-susceptible plants and is positively associated with glyphosate dose. These results suggest that under continued glyphosate selection pressure, the RR biotype may surpass the NRR biotype as the predominant form of GR giant ragweed in Indiana due to a higher level of glyphosate resistance. Moreover, the differential photo-oxidative stress patterns in response to glyphosate provide evidence of different mechanisms of resistance present in RR and NRR biotypes.