Cytogenetic and molecular investigations on the holocentric chromosomes of the aphid Macrosiphum euphorbiae (Thomas, 1878) have been carried out using silver staining and C-banding (followed by chromomycin A(3) and DAPI staining) in order to improve our knowledge about the structure of aphid chromosomes. The C-banding pattern is peculiar since only the two X chromosomes and a single pair of autosomes presented heterochromatic bands. Silver staining and FISH with the 28S rDNA probe localized the rDNA genes on one telomere of each X chromosome that were also brightly fluorescent after chromomycin A(3) staining of C-banded chromosomes, whereas all other heterochromatic bands were DAPI positive. Interestingly, a remarkable nucleolar organizing region (NOR) heteromorphism was present making the two X chromosomes easily distinguishable. Southern blotting and FISH assessed the presence of the (TTAGG)(n) repeat at the ends of all the M euphorbiae chromosomes. Karyotype analysis showed that all males possessed the X chromosome with the larger amount of rDNA suggesting a non-Mendelian inheritance of the two X chromosomes.