Landoltia was collected and cultured from a canal in Lake County, Florida, where diquat was used repeatedly during the past 20–30 yr for duckweed control. Recent applications of diquat failed to provide adequate control of duckweed, and a new commercial formulation of diquat was suspected. The new formulation was not the cause of reduced efficacy. Static exposures (48 h) to various concentrations of diquat were used to compare the susceptibility of the Lake County landoltia accession to one never exposed to diquat. These static tests indicated that landoltia, from a population with no prior history of herbicide treatment, was extremely susceptible to diquat. The accession from Lake County, FL had developed resistance to diquat, and was also cross resistant to paraquat. The resistance factor was 50 for diquat and 29 for paraquat. The Lake County accession also exhibited reduced ion leakage after diquat exposure under light and dark conditions. This suggests the resistance mechanism to the bipyridylium herbicides in landoltia is independent of photosynthetic electron transport. This research documents the first aquatic plant species that has developed resistance to the bipyridylium herbicides.