The present investigation was carried out on six economically important species of Asparagus using chromosomal characteristics, 4C nuclear DNA contents, SDS PAGE of total leaf proteins, isozyme polymorphism, and molecular markers to assess their relationship both at inter- and intraspecific levels. Karyotype analysis revealed diploid (2n=2x=20) status in most of the species except tetraploidy (2n=4x=40) in A. cooperi and hexaploidy (2n=6x=60) in A. densiflorus. Karyotype asymmetry existed among the species as well as in same ploidy level of different species. The inter-chromosomal and intra-chromosomal asymmetry ranged from values of 0.902-0.977 and 0.197-0.486, respectively. Again, considering chromosome number, length, volume, and median chromosome number, the primitive nature has been observed in A. densiflonis. The difference in chromosome parameters in all the species suggested the occurrence of structural changes at the chromosomal level. However, a higher degree of correlations has been found between chromosome volume and 4C nuclear DNA content in the six species studied so far. The dendogram obtained from SDS-PAGE and isozyme analysis clearly revealed a close relationship between A. racemosus and A. pyramidalis, but the dendogram obtained from RAPD and ISSR revealed a close relationship among A. pyramidalis, A. densiflorus, and A. racemosus. Asparagus plumosus and A. cooperi also showed a close relationship in all aspects except in ISSR study. All these data clearly indicated that a better phylogenetic relationship can be drawn from the combination of cytological and molecular marker-based analysis, which may assist the breeding pattern or germplasm conservation of the gene pool of specified crops. © 2013 The Japan Mendel Society.