Chromosomal mapping of 5S rDNA in two Acanthocephala species was performed for the first time using fluorescence in situ hybridization (FISH) with a 5S rDNA probe. The 5S rDNA PCR products from the genomes of both species were sequenced and aligned and an identical 141 bp long coding region was determined. The same patterns of 5S rDNA gene cluster distribution were observed, with FISH signal restricted to a single autosomal chromosome pair. A preference for distal positioning on the chromosomes (subtelomeric position) was observed, namely on the short arms of the submetacentric chromosome in Acanthocephalus lucii and on the short arms of the acrocentric chromosome in A. anguillae. In addition, two-color FISH was performed to examine the mutual positions of 5S and 18S rDNA on the chromosomes. Our knowledge of the organization of the Acanthocephala genome is extremely limited and its chromosomes are poorly studied. Any new information about the location of chromosomal markers as important features of the respective karyotype may be useful in solving evolutionary questions.