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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 A3 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 A3 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.