Recent genomic data suggest that the role of hybridization in evolution might be more important than previously assumed. Here, we examine species-specific differentiation and signatures of reticulation in a multi-species complex of the crustacean genus Daphnia. We did a combined examination of mtDNA, allozymes and ITS1 (a part of the nuclear ribosomal DNA) in the Daphnia longispina group. We focused on the sequence variation of ITS1 in two unambiguous species (D. galeata, D. cucullata) and two ecotypes hyalina and rosea within the recently revised taxon D. longispina (O.F. Müller). We found two ITS1-types (S, L) and intra-individual and intra-specific polymorphisms. Another ITS1-type (XL) was restricted to the outgroup D. umbra. S was present in all taxa but occurred as only two variants. Surprisingly, D. galeata and D. cucullata, which were well differentiated by mtDNA and allozymes, were virtually indistinguishable with respect to S-ITS1 (Scg). The two ecotypes of D. longispina shared the second S-ITS1-variant (Srh) and were therefore almost indistinguishable for all types of molecular markers surveyed. The L-type differed between D. galeata and D. longispina samples, but L was absent in D. cucullata. Between hyalina and rosea ecotypes, we found some L-differentiation. Combined data suggest that reticulate evolution enabled the spread of one S-ITS1-variant (Scg) beyond species boundaries and that S-introgression was species-specific, despite contemporary hybridization between all species. Our data have implications for phylogenetic as well as phylogeographic surveys. Because of the dynamic impact of gene flow in multi-species complexes, misinterpretations of presumed species-specific data should be considered. J. Exp. Zool. 311A:530–548, 2009. © 2009 Wiley-Liss, Inc.