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Ophichthidae is the most ecologically diverse family of the order Anguilliformes; however, high morphological similarity and a long larval stage make precise identification difficult. The aim of the present study was to examine the potential for barcoding of ophichthid fishes. A total of 81 mitochondrial cytochrome C oxidase subunit I (COI) barcode sequences were obtained from 23 species and 12 genera of ophichthid fishes collected in the Taiwan Strait. The average Kimura two-parameter distances within species, genera, and families were 0.48%, 18.25%, and 18.79%, respectively; thus, the mean interspecific distance was 31-fold higher than the mean intraspecific distance, which demonstrates good efficiency in DNA barcoding. The sequence divergence among species also varied substantially from 4.60% to 25.90%. This evaluation demonstrates the high efficiency of DNA barcoding for species identification to facilitate research on the diversity and conservation of ophichthid fishes.