Specimens of Contracaecum spp. from Phalacrocorax auritus and Pelecanus occidentalis from Florida were characterized by sequencing of the small subunit of the mitochondrial ribosomal RNA gene (rrnS) and by PCR-based RFLP analysis of the same gene and of the internal transcribed spacers (ITS) of nuclear ribosomal DNA. Analyses of the rrnS sequence data using the MP and UPGMA approaches yielded trees with similar topologies, delineating 3 main clusters. Specimens from Ph. auritus, morphologically assigned to C. rudolphii (s.l.), were part of the cluster comprising also the other 2 species of the C. rudolphii complex (A and B), but representing a genetically distinct group, potentially corresponding to a distinct lineage within the complex, provisionally named as C. rudolphii C. The second cluster comprised 5 individuals from P. occidentalis, which formed a genetically relatively homogeneous group. The rrnS data indicate that these specimens (indicated as Contracaecum sp. 1) are clearly genetically different from the morphologically most closely related species, i.e. C. rudolphii (s.l.) and C. microcephalum, and could represent a new species. The third cluster comprised 7 specimens from P. occidentalis morphologically assigned to C. multipapillatum (s.l.). These were shown to be genetically homogeneous and related to but quite distinct from C. multipapillatum from Greece, although additional studies are needed to assess their status. PCR-RFLP based markers for the quick identification of these taxa are provided.