BackgroundThe bacterial speck disease of tomato caused by a bacterial pathogenPseudomonas syringaepv.tomatois a most important disease causing severe crop losses.Methods and resultsPresent study was conducted to investigate and characterize the population diversity ofP. syringaepv.tomatopathogen isolated from infected tomato plants from various regions of Egypt. Significant variation among the isolates was observed which demonstrated considerable virulence. All isolates were pathogenic and the CFU population recovered from inoculate tomato leaves by isolate Pst-2 was higher than other isolates. Genetic disparity among the isolates was investigated by PCR analysis by amplifyinghrpZ gene using random amplified polymorphic DNA (RAPD), sequence-related amplified polymorphism (SRAP), and inter-simple sequence repeats (ISSR) markers. The amplified products for ITS1 were found to have 810 bp length whereas 536 bp length was observed forhrpZ gene using primer pairs (1406-f/23S-r) and (MM5-F, MM5-R) respectively. The restriction analysis of amplified regions “ITS” andhrpZby using 5 and 4 endonucleases respectively demonstrated slight variation among the bacterial isolates. The results of RAPD, ISSR and SRAP showed higher polymorphism (60.52%) within the isolates which may assist for successful characterization by unique and specific markers based on geographical distribution, origin and virulence intensity.ConclusionThe results of present study suggested that the use of molecular approach may provide successful and valuable information to differentiate and classifyP. syringaepv.tomatostrains in future for the detection and confirmation of pathogenicity.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11033-023-08302-x.