Ten species (13 accessions) of the genus Asparagus were analysed to reveal their genome size structure and genetic similarity. The choice of species was based on their possible application in crossbreeding with Asparagus officinalis L. and their agricultural value . Flow cytometric measurement of genome size revealed two distinctive groups, based on 1C values, the European dioecious species having on average twice the genome size of the South African hermaphrodite species. Variability among species was relatively high both as absolute values (from 1.43 to 7.87 pg per nucleus) or expressed as 1C values (0.67–1.71 pg/1C). Additionally, Asparagus maritimus was determined as hexaploid in contrast to previous reports of it being tetraploid. Based on restriction fragment analysis of internal transcribed spacers (ITSs) of nuclear ribosomal DNA, the same two, distinctly distant groups of species were formed, which clearly separate species by their gender and geographic origin. The clustering obtained by the UPGMA method is in agreement with some botanical classifications. The genetic similarity and ploidy level results explain the successful interspecific hybridisation reported for A. officinalis and Asparagus tenuifolius and the failure of hybridisation between A. officinalis and some other Asparagus species.