The somatic chromosome numbers 2n = 12 in S. macrantha, S. coerulescens and 2n = 24 in S. simplicuscula were determined, additionally the contradictory chromosome numbers of S. bispinosa (2n = 12, 13, 14, 24) and S. pachycarpa (2n 12, 14) were determined as 2n = 24 and 2n = 14, respectively. The number of 5S rDNA sites in chromosome pair 1 was highly conserved in all the diploid and tetraploid species studied irrespective of their geographic distribution, suggesting that all diploid and tetraploid species/cytotypes of Sesbania analyzed in the present study are in close proximity. Cytogenetic mapping of the 45S multi-gene family was also carried out using fluorescence in situ hybridization. 45S rDNA was consistently located on short or long arms of two sub-metacentric chromosome pairs except on one chromosome pair in S. macrantha and on three chromosome pairs in S. bispinosa and S. cannabina. Out of these nine species, we observed the homogenization of intergenic spacer in six species and find only one spacer length variant (slv) located on one to three chromosome loci. However, three of the species were observed to have two slvs located on two different chromosomes. The species were grouped as per their evolutionary relationship on the basis of the results of the present study.