Marine free-living nematodes (Phylum Nematoda) are widespread and abundant in marine sediments, often representing 70%-90% of the benthic metazoans. However, marine nematode taxonomy is severely underdeveloped, and about only 4,000 species of free-living marine nematodes have been described. Nematode identification by using traditional morphological methods is time consuming and expensive. Some marine nematodes are small, displaying similar morphological characters and are difficult to identify by traditional methods. Molecular technology, or "barcoding," offers the potential of a fast and objective way of species identification. The small and large subunit ribosomal DNA (SSU rDNA and LSU rDNA) gene-based phylogenetic analysis is a powerful tool for clarifying evolutionary relationships among nematode taxa. The order Enoplida is one of the most important groups of marine nematodes. Many enoplids are possibly active predators, and play important ecological roles in marine environments. Here we reported the isolation of 26 nematodes, belonging to Enoplida, from sediment samples collected at four sites in the Clarion-Clipperton Fracture Zone of the Pacific Ocean. The specimens were preserved in DESS solution (20% dimethyl sulphoxide, 0.25 mol/L disodium EDTA pH 8.0, saturated with NaCl) immediately after collection. Each sediment sample was rinsed through a 38-μm sieve using filtered seawater, and extracted using the Ludox flotation method. Nematodes were placed on temporary slides and observed using a Leica DM5500 microscope. After image capturing, each specimen was washed, cut into several pieces, transferred into micro-centrifuge tubes, and digested with Proteinase K. A series of frozen specimens was subsequently thawed and subjected to PCR amplification of the 18S rRNA gene and D3 expansion segments of the 28S rRNA gene. Sequences were analyzed and compared with published data from GenBank by means of a BLAST search. Phylogenetic trees were constructed using the neighbor-joining, maximum likelihood, and maximum parsimony methods with the MEGA5 program package, after multiple alignment of the data by CLUSTAL W. Based on morphological and molecular analyses, these 26 nematodes were classified into six families and eight genera, among which Oxystominidae was the most abundant family, accounting for 57.7%. The other families included Anticomidae (19.2%), Phanodermatidae (7.7%), Oncholaimidae (7.7%), Ironidae (3.85%), and Enchelidiidae (3.85%). At the family and genus level, the community composition at adjacent sites during the same period showed similar results, but the abundance was different. Sixteen sequences of rRNA gene were obtained in the present study and their similarity to the sequences in GenBank ranged from 94 to 99%. According to the results from BLAST, all sequences could be identified at the level of family, while 84.6% of the 16 sequences could be identified at the level of genus. The results from morphological and molecular analysis showed high consistency, which suggested that molecular barcoding is an efficient method to identify deep-sea nematodes. Phylogenetic trees constructed from the sequences of 18S and 28S rRNA gene showed similar topological structures; the species of Oncholaimidae and Enchelidiidae were clustered into one group, whereas those of Phanodermatidae and Anticomidae were clustered into another group, indicating their close genetic relationships.