BACKGROUND: Anguillicolidae Yamaguti, 1935 is a family of nematode parasites infecting fresh-water eels of the genus Anguilla, with five species in the genera Anguillicola and Anguillicoloides. Anguillicolidae is part of Spirurina, a diverse clade made up of only animal parasites. Anguillicoloides crassus is of particular importance, as it has recently spread from its endemic range in the Eastern Pacific to Europe and North America, where it poses a significant threat to new, naïve hosts such as the economic important eel species Anguilla anguilla and Anguilla rostrata. The Anguillicolidae are therefore all potentially invasive taxa, but the relationships of the described species remain unclear. RESULTS: We generated an extensive DNA sequence dataset from three loci (the 5' one-third of the nuclear small subunit ribosomal RNA, the D2-D3 region of the nuclear large subunit ribosomal RNA and the 5' half of the mitochondrial cytochrome c oxidase I gene) for the five species of Anguillicolidae and used this to investigate species and generic boundaries within the family, and the relationship of Anguillicolidae to other spirurine nematodes. Neither nuclear nor mitochondrial sequences supported monophyly of the genera Anguillicola and Anguillicoloides. Genetic diversity within the African species Anguillicoloides papernai was suggestive of cryptic taxa, as was the finding of divergent lineages of Anguillicoloides novaezelandiae in New Zealand and Tasmania. Phylogenetic analysis of the Spirurina grouped the Anguillicolidae together with members of the Gnathostomatidae and Seuratidae. CONCLUSIONS: The Anguillicolidae is part of a complex radiation of vertebrate parasitic nematodes with wide host diversity (cartilaginous and bony fish, reptiles and mammals), most closely related to other parasites of marine vertebrates that also have complex lifecycles. Molecular analyses do not support the recent division of Anguillicolidae into two genera. The described species may hide cryptic taxa, identified here by DNA taxonomy, and this DNA barcoding approach may assist in tracking species invasions. The propensity for host switching, and thus the potential for invasive behaviour, is found in A. crassus, A. novaezelandiae and A. papernai, and thus may be common to the group.