The Swiss Barcode of Life initiative (SwissBOL) aims to inventory the genetic biodiversity in Switzerland using a short DNA sequence. DNA barcoding provides an additional tool for species identification that complements traditional morphological approaches. We report on the establishment of a DNA barcode library for Plecoptera, taxa that are of great importance as bioindicators of water quality and that often present difficulties in species-level identification for larvae and female specimens. Non-destructive DNA extraction, PCR amplification and sequencing of part of the mitochondrial gene Cytochrome Oxidase I (COI) was conducted for 440 individuals (one to eight per species) belonging to 90 species (of the 112 reported from Switzerland). Intra and interspecific distances were calculated and gene trees reconstructed. In most cases, COI was efficient in delimiting stonefly species. Some doubtful specimens were subsequently re-examined and a few misidentifications were found, especially in some problematic groups in the genus Leuctra Stephens, 1836. Larger genetic distances in some species (e.g. Leuctra nigra (Olivier 1811)) indicate the possible presence of sibling species, while in a few cases closely related species are genetically difficult to separate (within the Leuctra fusca species group).