The diversity and phylogenetic relationships of the Desmodoridae, a widespread tropical family of free-living marine nematodes, is hitherto poorly known both from molecular and taxonomic points of view. We performed a molecular phylogenetic analysis of marine nematodes to: i) disentangle relationships among tropical desmodorid species; and ii) compare the performance of the nuclear SSU rDNA and mitochondrial COI nucleotide sequences in 42 and 45 nominal species, respectively, to identify species. We generated 27 new sequences of SSU rDNA belonging to five genera not previously sequenced, and 34 new sequences of COI belonging to six genera and four families not previously sequenced. The SSU rDNA tree confirmed the Enoplida to be a monophyletic sister group to the Chromadorida. The family Comesomatidae is a sister group of the Xyalidae within the Monhysterida. Both DNA markers confirmed the congruence between the morphology- and molecular-based phylogenetic inferences for most of the families. Desmodoridae was a monophyletic group, but the relationships within the family could not be recovered; the subfamilies Desmodorinae and Spiriniinae were not monophyletic meanwhile the monophyly of Stilbonematinae was not fully supported due to a few specimens of questionable identity. COI performed better than SSU rDNA to disentangle relationships among closely related species and suggested the presence of cryptic diversity within Desmodoridae. COI is effective to explore cryptic diversity and barcode species within Nematoda, with a possible threshold of genetic distance of 5% between conspecific and interspecific sequences, but DNA barcoding is limited by the poor knowledge of the diversity and taxonomy of the group and the lack of a good reference database of vouchered COI sequences.