One of the fundamental aspects of evolutionary biology is understanding genome organization and its functional aspects that directly or indirectly act in concert with the adaptability of any organism. The evolution of chromosome size, number, and structure, as well as changes in DNA composition, suggest the high plasticity of nuclear genomes at the chromosomal level. Hence, chromosomal information on chromosomal complements bears significant importance in plant breeding and genetic studies, including genome analysis. Regardless of the prime focus on the mulberry crop, the understanding of its ploidy variation and ploidy-associated traits is lacking, which hinders the development of strategies for its systematic improvement and conservation. In the present research, with the execution of classical cytology, the chromosome numbers of 20 mulberry accessions (Morus spp.) have been estimated using shoot tip meristems. Chromosomal complements accounted for definitive intraspecific variation at ploidy levels such as diploid (2n=2x=28), triploid (2n=3x=42), tetraploid (2n=4x=56), and hexaploid (2n=6x=84) among the studied species (M. alba, M. australis, M. bombycis, M. cathayana, M. indica, M. laevigata, M. latifolia, M. macroura, M. rotundiloba, and M. serrata). Additionally, flow cytometry analysis was performed for the validation of chromosomal data. To identify ploidy-associated traits, different functional traits were studied. Principal component analysis (PCA) and heat map analysis indicate the positive impact of genome duplication on leaf anatomical traits. Further consideration of below-ground traits will require in-depth understanding. Moreover, the present outcomes of intraspecific ploidy variation and identified ploidy-associated traits provide a platform and open windows for frontier omics approaches to improve the mulberry crop in the near future.