Florida hydrilla populations have shown an alarming increase in resistance to fluridone, an herbicide used extensively for controlling invasive US hydrilla populations. A rapid PCR and sequencing method was developed to identify and screen hydrilla genomic DNA for three previously identified phytoene desaturase (pds) gene mutations that confer resistance to fluridone. Ninety hydrilla accessions were screened for fluridone resistant genotypes including 46 accessions from the US and 44 accessions from 15 other countries. In Florida, hydrilla from five of nine sites tested was heterozygous for wild-type and herbicide-resistant alleles. Additionally, a new resistant population was identified from Lake Seminole in Georgia, the first genetically confirmed strain of resistant hydrilla outside of Florida. All resistance-conferring mutations were located on the same homologous haplotype of US dioecious hydrilla. All other hydrilla samples tested possessed only wild type alleles, including monoecious strains that had been exposed to fluridone. Management implications are discussed.