Detecting and diagnosing seed-borne bacterial pathogens is imperative for mitigating their detrimental impact on the market value and yield of tomatoes, as well as for preventing potential epidemics. In this research, 50 seed samples originating from 30 distinct tomato cultivars/genotypes across 10 provinces in 6 regions of Turkey were gathered from commercial entities or local producers. The presence of bacterial pathogens within the seeds was ascertained through the implementation of standard ISTA test protocols, encompassing biochemical, morphological, physiological, and molecular assessments. The findings revealed the presence of 10 highly pathogenic strains of Xanthomonas vesicatoria (Xv) and 9 strains of Pseudomonas syringae pv. tomato (Pst) with occurrence rates of 18.8% and 16.36%, respectively, among all seed samples. Seven ISSR markers and nine SRAP markers were employed to assess the genetic disparities among Xv and Pst strains categorized as pathogens originating from different climatic regions. Detecting seed-borne bacterial pathogens in extensively cultivated tomatoes and elucidating the genetic diversity among them would empower producers to cultivate cultivars tailored with desired traits, particularly disease-resistant attributes.