The present paper reports on a first attempt at resolving the taxonomy of Chilean Pycnogonida using a combination of DNA sequence and morphological data. In a subproject of the Marine Barcode of Life (MarBoL) campaign we analysed a fragment (about 657 base pairs) of the mitochondrial protein-coding gene COI (cytochrome c oxidase subunit 1) from 76 Chilean/Subantarctic pycnogonids. Since most molecular data on pycnogonids are from the Antarctic region, the new information constitutes a significant extension. The phylogenetic consensus tree displays 10 distinct, well-supported branches corresponding to the studied species, namely Achelia assimilis (Haswell, 1885), Ammothea spinosa (Hodgson, 1907), Tanystylum cavidorsum Stock, 1957, T. neorhetum Marcus, 1940, Colossendeis macerrima Wilson, 1881, C. megalonyx Hoek, 1881, C. scoresbii Gordon, 1932, Callipallene margarita (Gordon, 1932), Pallenopsis patagonica (Hoek, 1881), and Anoplodactylus californicus Hall, 1912. These represent four superfamilies, and five of the 11 existing pycnogonid families (Bamber & El Nagar, 2011): Ammotheidae Dohrn, 1881, Colossendeidae Hoek, 1881, Callipallenidae Hilton, 1942, Pallenopsidae Fry, 1978 and Phoxichilidiidae Sars, 1891. Within Achelia assimilis, four distinct subbranches correspond to the different geographic regions represented in our samples. While these include a total of 11 distinct haplotypes, the morphological differences among the corresponding specimens lie well within the variation described in the literature for this cosmopolitan species. Therefore, the four branches of A. assimilis might represent geographically limited subspecies rather than cryptic species. Repeated drastic glaciation of the fjord region during the Cenozoic resulting in alternating extinction and recolonization phases and the holobenthic lifecycle of sea spiders are discussed as the main factors resulting in the observed phylogeographic pattern. Standard barcoding sequences are confirmed as a suitable tool in addition to morphology for taxonomic analyses in Pycnogonida. The corresponding haplotype distribution patterns allow inferences on the biogeographical history of the relatively unexplored Chilean fjord region.