Polymerase chain reaction primers based on the ureC gene are described for use in detecting diverse groundwater urea-hydrolyzing bacteria. Six degenerate primers were designed and evaluated for their ability to detect the gene encoding the large catalytic subunit of urease, ureC. Five combinations of these primers were tested pair-wise and displayed an overlapping detection range for bacterial isolates. Pair L2F/L2R exhibited the greatest detection range for described bacterial species and for bacterial isolates from groundwater samples belonging to the bacterial divisions Firmicutes, Actinobacteria, and the , β, and γ subdivisions of Proteobacteria. Primers L2F/L2R exhibited a greater detection range than previously described ureC-specific primers, and amplified novel ureC sequences from groundwater isolates in the genera Hydrogenophaga, Acidovorax, Janthinobacterium, and Arthrobacter. A comparative phylogenetic analysis of ureC and 16S rRNA genes was performed to determine the utility of groundwater ureC sequence information as a phylogenetic marker for ureolytic species. Our results were consistent with previous analyses of urease genes which demonstrated that the ureC gene has undergone lateral transfer and is not a robust phylogenetic marker. However, the ureC-specific primers, L2F/L2R, demonstrate a broad detection range for ureolytic species, and can serve to enhance functional diversity analyses of ureolytic bacteria.