In this study, we performed a cytogenetic analysis of Acanthocephalus lucii specimens from three sites with different levels of environmental pollution. Standard and fluorochrome staining (CMA3/DAPI), fluorescence in situ hybridization (FISH) with 18S rDNA and histone H3 probes, and silver impregnation were performed. Chromosome complements of 2n = 7/8 (male/female), n = 1m + 2sm + 1a (X), and CMA3-positive bands in all chromosomes were found in all three populations. FISH revealed one 18S rDNA locus on the X chromosome and one locus of H3 histone genes on the first chromosome pair. At the intraspecific level, the populations differed in the presence of supernumerary B chromosomes, which were found in all specimens from Zemplínska Šírava and in 89.4% of specimens from the Laborec River, but not at the reference site. The first two sites are considered to be water bodies with high toxin contamination. Based on this fact, we assume an increased frequency of chromosome breaks leading to the formation of DNA fragments that have the potential to form B chromosomes. The present results add to the very limited data on the organization of multigene families in the genome of Acanthocephala and suggest a possible causal link between water pollution and the occurrence of B chromosomes in fish parasites.