Three Brassica species, namely, Brassica rapa, B. nigra, and B. oleracea are considered economically important as they are grown for human consumption and biogas production. Like other crops facing agricultural constraints, selective crossing or hybridization in cruciferous vegetables has helped farmers to improve them. This study conducted a comparative evaluation across and within the species through cytogenetic analysis to provide fresh insights into their chromosome structures and evolutionary relationships. A new karyomorphological parameter confirmed symmetric karyotypes in all the accessions, thereby allowing for the depiction of the ancestral chromosome karyotype. Several lines of B. rapa, B. nigra, and B. oleracea were subjected to physical mapping using a fluorescence in situ hybridization technique to elucidate the chromosomal distribution of the two types of rDNAs. The signal number and distribution of 18S rDNA across the metaphase chromosomes of B. rapa accessions did not vary as compared to 5S rDNA, which was also observed in several lines of B. nigra. In contrast, the number and distribution of 5S rDNA loci across the chromosomes in several lines of B. oleracea were found to be more conserved than those corresponding to the 18S rDNA. Overall, this study revealed the evolutionary dynamics of rDNA, which may play an important role in shaping the chromosome karyotypes of Brassica species.