DNA sequence data and phylogenies are useful tools for species delimitation, especially in taxa comprising cryptic species. The Lepidocyrtus lanuginosus species group (Collembola: Entomobryidae) comprises three morphospecies and distinct cryptic species. We applied three DNA-based methods to delimit species boundaries in the L. lanuginosus species group across central and southern Europe. Using cytochrome c oxidase subunit I and II, we identified gaps of genetic distances that indicate species boundaries and found 10 and 9 distinct genetic lineages in L. cyaneus and L. lanuginosus, respectively. The nuclear gene elongation factor 1-α delimited 89% of the lineages but 28S rDNA (D1–2 domain) was too conserved for this purpose. The phylogenetic trees showed that L. cyaneus and L. lanuginosus are polyphyletic, suggesting that body colour is insufficient for delimiting species in the L. lanuginosus species group. Our study challenges the current morphology-based species delimitation in the L. lanuginosus species group and suggests that molecular approaches are needed for fast and accurate determination of Collembola species in both taxonomic and ecological studies. Overall, the results suggest that wide geographic sampling combined with molecular phylogenetic approaches is needed to delimit species and to understand the full range of cryptic diversity in Collembola.