Applicability of the polymerase chain reaction (PCR) with species-specific primers to obtaining molecular markers for identification of the sibling species of Chironomus plumosus group - C. plumosus and C. balatonicus - has been estimated. The nucleotide sequences of internal transcribed spacer (ITS) from the locus encoding ribosomal RNA (rRNA) were used as the source for designing the species-specific primers. The primers allowing for identification of C. plumosus and C. balatonicus were constructed. One primer pair (plu107F/plu363R) gives the PCR product MAR2, specific of C. plumosus, and the other (bal86F/plu363R), the PCR product MAR6, specific of C. balatonicus. The testing involving 18 species of the genus Chironomus confirmed the specificity of the primers. The results suggest that the PCR with species-specific primers is promising for construction of molecular markers for identification not only of these two, but also of other Chironomus species. doi: 10.5324/fn.v31i0.1381. Published online: 17 October 2012.