Understanding animal foraging ecology requires large samples sizes spanning broad environmental and temporal gradients. For pollinators, this has been hampered by the laborious nature of morphologically identifying pollen. Metagenetic pollen analysis is a solution to this issue, but the field has struggled with poor quantitative performance. Building upon prior laboratory and bioinformatic methods, we applied quantitative multi-locus metabarcoding to characterize the foraging ecology of honey bee colonies situated along an urban-agricultural gradient in central Ohio, USA. In cross-validating a subset of our metabarcoding results using microscopic palynology, we find strong concordance between the molecular and microscopic methods. Our results show that, relative to the agricultural environment, urban and suburban environments were associated with higher taxonomic diversity and temporal turnover of honey bee pollen forage. This is likely reflective of the fine-grain heterogeneity and high beta diversity of urban floral landscapes at the scale of honey bee foraging. Our work also demonstrates the power of honey bees as environmental samplers of floral community composition at large spatial scales, aiding in the distinction of taxa characteristically associated with urban or agricultural land use from those distributed ubiquitously across our landscape gradient.