Urea is an important and dynamic natural component of marine nitrogen cycling and also a major contributor to anthropogenic eutrophication of coastal ecosystems, yet little is known about the identities or diversity of ureolytic marine microorganisms. Primers targeting the gene encoding urease were used to PCR-amplify, clone and sequence 709 urease gene fragments from 31 plankton samples collected at both estuarine and open-ocean locations. Two hundred and eighty-six amplicons belonged to 22 distinct sequence types that were closely enough related to named organisms to be identified, and included urease sequences both from typical marine planktonic organisms and from bacteria usually associated with terrestrial habitats. The remaining 423 amplicons were not closely enough related to named organisms to be identified, and belonged to 96 distinct sequence types of which 43 types were found in two or more different samples. The distributions of unidentified urease sequence types suggested that some represented truly marine microorganisms while others reflected terrestrial inputs to low-salinity estuarine areas. The urease primers revealed this great diversity of ureolytic organisms because they were able to amplify many previously unknown, environmentally relevant urease genes, and they will support new approaches for exploring the role of urea in marine ecosystems.