Asparagus wild relatives could be a promising possibility to extent the genetic variability of garden asparagus and for new cultivars with favorable traits such as high yield stability, disease resistance and stress tolerance. In order to achieve an efficient use in breeding, a detailed cytogenetic characterization of the accessions is necessary. This study worked on 35 Asparagus accessions, including A. officinalis cultivars ('Darlise', 'Ravel' and 'Steiners Violetta') and Asparagus wild relatives, for which the number of chromosomes, their size, the nuclear DNA content, and the genomic distribution of 5S and 45S rDNA were analyzed. Different ploidy levels (diploid, triploid, tetraploid, pentaploid and hexaploid) were found. Furthermore, the size of the chromosomes of all diploid Asparagus accessions was determined which led to differences in the karyotypic formula. A. plocamoides harbors the smallest chromosome with 1.21 μm, whereas the largest chromosome with 5.43 μm was found in A. officinalis. In all accessions one 5S rDNA locus per genome was observed, while the number of 45S rDNA loci varied between one (A. albus, A. plumosus, A. stipularis) to four (A. setaceus). In most Asparagus accessions, the 5S and 45S rDNA signals were located on different chromosomes. In contrast, the genomes of A. africanus, A. plocamoides, A. sp. (a taxonomically unclassified Asparagus species from Asia) and A. verticillatus (diploid accessions) have one 5S and one 45S rDNA signal on the same chromosome. The measured 2C DNA content ranges from 1.43 pg (A. plocamoides, diploid) to 8.24 pg (A. amarus, hexaploid). Intraspecific variations for chromosome number, karyotypic formula, signal pattern with 5S and 45s rDNA probes and DNA content were observed. Interspecific variations were also recognized in the genus Asparagus.