Karyotype and cytogenetic characteristics of critically endangered Iberian endemic cyprinid fish Parachrondrostoma arrigonis (Steindachner, 1866) were investigated using sequential C-banding, Ag- and CMA3-stainings in two specimens identified on morphological criteria. The diploid chromosome number was 2n = 50 with 8 pairs of metacentrics, 14 pairs of submetacentrics to subtelocentrics and 3 pairs of subtelo-acrocentrics. The largest chromosome pair of the complement was characteristically subtelo- to acrocentric. In the single analysed male, the nucleolar organizer regions (NORs) detected by Ag- and CMA3-stainings were in the telomeres of a single, middle-sized subtelocentric chromosome pair which is a pattern common in a number of other Leuciscinae. In the single analysed female, in which we could not confirm or reject interspecific hybridization, the NOR phenotype detected by these stainings showed at least seven positive sites on various chromosomes including characteristic large subtelo- to acrocentric pair of chromosomes. The CMA3-fluorescence revealed also in this female an entirely CMA3-positive arm, nature of such phenomenon (variation) is unknown at present. The complicated NOR phenotype of this specimen might suggest either hybrid situation or possible involvement of an association between mobile elements and rDNA loci. Karyotype structure homogeneity found for male of P. arrigonis with those in the most other representatives of the European leuciscine fishes may confirm karyotypic conservation within this cyprinid lineage.