Brittle stars (Echinodermata: Ophiuroidea) comprise over 2,000 species, all of which inhabit marine environments and can be abundant in the deep sea. Morphological plasticity in number and shape of skeletal parts, as well as variable colors, can complicate correct species identification. Consequently, DNA sequence analysis can play an important role in species identification. In this study we compared the genetic variability of the mitochondrial cytochrome c subunit I gene (COI) and the nuclear small subunit ribosomal DNA (SSU, 18S rDNA) to morphological identification of 66 specimens of 11 species collected from the North Atlantic in Icelandic waters. Also two species delimitation tools, Automatic Barcode Gap Discovery (ABGD) and General Mixed Yule Coalescence Method (GMYC) were performed to test species hypotheses. The analysis of both gene fragments was successful to discriminate between species and provided new insights into some morphological species hypothesis. Although less divergent than COI, it is helpful to use the SSU region as a complementary fragment to the barcoding gene.