The identification of spider species presents many challenges, since in most cases the characters used are from genital structures that are only fully developed in the adult stage, hence the identification of immatures is most often not possible. Additionally, these structures usually also present some intra-specific variability, which in some cases makes the identification of closely related species difficult. The genetic barcode technique (DNA barcodes), based on sequencing of the mitochondrial marker cytochrome c oxidase subunit I (COI), has proven a useful, complementary tool to overcome these limitations. In this work, the contribution of DNA barcoding to the taxonomy of the subfamily Amaurobioidinae is explored using the refined single linkage analysis (RESL) algorithm for the delimitation of operational taxonomic units (OTUs), in comparison with the assemble species by automatic partitioning (ASAP) algorithm, and presented in conjunction with an updated molecular phylogenetic analysis of three other markers (28S rRNA, 16S rRNA, Histone H3), in addition to COI. Of a total of 97 included species identified by morphology, 82 species were concordant with the operational taxonomic units obtained from RESL, representing an 85% correspondence between the two methods. Similar results were obtained using the ASAP algorithm. Previous observations of morphological variation within the same species are supported, and this technique provides new information on genetic structure and potentially cryptic species. Most of the discrepancies between DNA barcoding and morphological identification are explained by low geographic sampling or by divergent or geographically structured lineages. After the addition of many specimens with only COI data, the multi-marker phylogenetic analysis is consistent with previous results and the support is improved. The markers COI, closely followed by 28S, are the most phylogenetically informative. We conclude that the barcode DNA technique is a valuable source of data for the delimitation of species of Amaurobioidinae, in conjunction with morphological and geographic data, and it is also useful for the detection of cases that require a more detailed and meticulous study.