Enhancing cotton yield and fiber quality is challenging due to the limited genetic variation, highlighting the need for efficient use of germplasm resources. Therefore, 10 morpho-physiological characters and 10 simple sequence repeat (SSR) markers were used to investigate the genetic variability of 50 cotton genotypes. Principal component analysis and Mahalanobis' generalized distance (D2) were applied to morpho-physiological data. Two principal components presented 53.83% of the cumulative variability in the raw data on biomass-related variables. Four different clusters were exposed by cluster analysis (D2): Cluster I included 20 genotypes, whereas clusters II, III, and IV had 6, 18, and 6 genotypes, respectively. Intra-cluster distances were highest in Cluster IV (222.79) and Cluster I (213.65), while Cluster III had the lowest (124.89), indicating greater genetic similarity. The largest inter-cluster distance was among Clusters II and IV (109.28), whereas the smallest was among Clusters II and III (20.67). A greater inter-cluster than intra-cluster distance suggests substantial genetic diversity among genotypes. Molecular-based explorations of genetic variability generated a matrix of similarity coefficients and grouped the accessions into two main clusters. Ten markers revealed polymorphism and produced a total of 35 clearly identifiable bands, with an average of 3.5 alleles per marker. In this study, the highest number of alleles (6) were amplified by DPL0009. Polymorphism Information Content values ranged from 0.076 to 0.731. Clustering based on molecular data classified the germplasm into three groups and measuring genetic distance through Euclidean distance while structure analysis revealed three gene-exchanging populations, representing a limited genetic basis in breeding programs. DNA clustering of upland cotton germplasm revealed genetic relationships independent of geographical origin, indicating its diversity and potential for use in hybridization schemes to develop superior F1 transgressive segregates or hybrids in subsequent generations.