<p>Integrating anatomical data with trustworthy molecular markers is necessary to comprehend taxonomic relationships within the family Rutaceae, especially for closely related farmed plants. Using leaf morpho-anatomical characteristics in conjunction with Start Codon Targeted (SCoT) and Sequence-Related Amplified Polymorphism (SRAP) markers, this study sought to assess the genetic diversity and taxonomic differentiation among sixteen Rutaceae taxa (11 genera) collected from the Aswan Botanical Garden (2023–2024). The diagnostic relevance of leaf epidermal and internal structural analysis for taxonomic separation within Rutaceae was supported by the clear interspecific variation seen in these analyses. Quantitatively, many features, including vascular bundle characteristics, epidermis thickness, and mesophyll organization, demonstrated consistent patterns that differentiated main lineages. The genetic links between taxa were further elucidated by molecular profiling. Five SRAP primer combinations provided 12 bands with a significantly greater polymorphism level of 70%, while seven SCoT primer combinations produced 18 reproducible bands, 32.14% of which were polymorphic. This disparity was emphasized by genetic diversity indices: Compared to SCoT, which produced PIC values of 0.25 and MI of 0.46, SRAP produced an average PIC of 0.37 and MI of 0.68. The sixteen taxa were consistently divided into two primary groups by cluster analysis based on individual and combined datasets, which mirrored patterns found in the anatomical results. Together, our results show that although both marker systems can detect genetic diversity, SRAP provided higher discriminatory power among Rutaceae taxa, while SCoT provided additional resolution as a complementary marker. A strong framework for taxonomic clarification, germplasm characterization and marker-based conservation strategies for Rutaceae in Egypt is provided by combining anatomical diagnostics with the two marker types.</p>