Onychophorans (velvet worms) use an adhesive, protein-based slime secretion for prey capture and defence. The glue-like slime is ejected via a pair of modified limbs and the sticky threads entangle the victim. In this study, we analysed the protein composition of slime in twelve species of Onychophora from different parts of the world, including two species of Peripatidae from Costa Rica and Brazil and ten species of Peripatopsidae from Australia, using sodium dodecyl sulphate polyacrylamide gel electrophoresis. Our results revealed high intraspecific conservation in protein composition of slime in each species studied. In contrast, the protein profiles differ considerably in both number and position of bands between the species. We observed the highest number of differences (in 20 of 33 considered band positions) between a peripatid and a peripatopsid species, whereas the lowest number of differences (in four band positions) occurs between two closely related egg-laying species. The reconstructed maximum parsimony cladogram based on the electrophoretic characters largely reflects the phylogenetic relationships of the species studied, suggesting that the slime protein profiles contain useful phylogenetic information. Based on our findings, we suggest that the slime protein profiling is a valuable, non-invasive method for identifying the onychophoran species. Moreover, this method might help to discover potentially new species of Onychophora, given that the ~200 described species most likely underrepresent the actual diversity of the group.