Leafy spurge, a noxious perennial weed, is a major threat to the prairie ecosystem in North America. Strategic planning to control leafy spurge requires monitoring its spatial distribution and spread. The ability to detect flowering leafy spurge at two biological control sites in southern Saskatchewan, Canada, was investigated using an unmanned aerial vehicle (UAV) system. Three flight missions were conducted on June 30, 2016, during the leafy spurge flowering period. Imagery was acquired at four flight heights and one or two acquisition times, depending on the site. The sites were reflown on June 28, 2017, to evaluate the change in flowering leafy spurge over time. Mixture tuned matched filtering (MTMF) and hue, intensity, and saturation (HIS) threshold analyses were used to determine flowering leafy spurge cover. Flight height of 30 m was optimal; the strongest relationships between UAV and ground estimates of leafy spurge cover (r2 = 0.76 to 0.90; normalized root mean square error [NRMSE] = 0.10 to 0.13) and stem density (r2 = 0.72 to 0.75) were observed. Detection was not significantly affected by the image analysis method (P > 0.05). Flowering leafy spurge cover estimates were similar using HIS (1.9% to 14.8%) and MTMF (2.1% to 10.3%) and agreed with the ground estimates (using HIS: r2 = 0.64 to 0.93, NRMSE = 0.08 to 0.25; using MTMF: r2 = 0.64 to 0.90, NRMSE = 0.10 to 0.27). The reduction in flowering leafy spurge cover between 2016 and 2017 detected using UAV images and HIS (8.1% at site 1 and 2.7% at site 2) was consistent with that based on ground digital photographs (10% at site 1 and 1.8% at site 2). UAV imagery is a useful tool for accurately detecting flowering leafy spurge and could be used for routine monitoring purposes in a biological control program.