This study aims to assess the genetic variability and heritability of key agronomic traits in 31 Cissus quadrangularis accessions, providing insights for breeding and conservation. Utilizing Randomized Complete Block Design (RCBD), plants were cultivated at Tamil Nadu Agricultural University, Coimbatore. Comprehensive statistical analyses were undertaken to elucidate trait relationships among the genotypes, including heritability, genetic variability, principal component analysis (PCA), and path analysis. Heritability estimates revealed high genetic control over traits, notably whole plant weight (WPW), with a heritability of 98.72%. Genotypic coefficients of variation (GCV) for leaf width and mature leaves per plant indicated significant genetic diversity, reinforcing their selection potential. PCA highlighted that the first two components explained 43.36% of the variance, with PC1 (25.51% variance) being primarily influenced by leaf width (LW), internodal length (IL), and whole plant weight (WPW), and PC2 (17.85% variance) being influenced by leaf length (LL) and petiole length (PL), guiding researchers in data reduction and trait prioritization. PC1 and PC2 highlight key traits for Cissus quadrangularis improvement. Path analysis demonstrated various traits' direct and indirect effects on plant growth, identifying leaf width as a critical influencer. Correlation analysis provided insights into trait interdependencies, with strong positive correlations observed between leaf width and internodal length. The phenotypic correlations, though generally weaker than genotypic, underscored the consistent expression of traits in different environments. Variance component analysis further established that traits like WPW and mature leaves per plant are predominantly governed by genetic factors, suggesting their potential for breeding programs. This research confirms genetic variability in Cissus quadrangularis and its medicinal potential, aiding trait selection in breeding programs. By integrating traditional knowledge with genetic analysis, this study provides a foundation for breeding strategies to enhance cultivation and medicinal utilization of Cissus quadrangularis.