Erwinia carotovora subsp. atroseptica (Eca), the causes of blackleg of potato, is closely resembles to E. carotovora subsp. carotovora (Ecc), the causes of bacterial soft rot of potato, in many physiological and biochemical characteristics. Nine isolates were obtained from potato tubers collected from different localities of El-Minia, Assiut and Sohag Governorates and subjected to this investigation. Three different molecular techniques were employed to differentiate the two subsp. of Erwinia carotovora. The primers used in RAPD-PCR technique generated an unique distinct bands which could be used as genetic markers to distinguish the isolates in respect of their sub species (carotovora or atroseptica) or their virulence (high or low virulence). Also, the 16s rRNA gene sequences was obtained from the bacterial isolates of Ecc and Eca. The phylogenetic tree couldn`t distinguish between the two subsp. of E. carotovora, where some isolates of Ecc connected in the same cluster with Eca. Moreover, SDS-PAGE analysis technique was used. The application of protein (SDS-PAGE) analysis may aid to detect the differences between the isolates of E. c. subsp. carotovora or atroseptica in the base of the region of collection. Where, protein analysis showed polypeptide fractions could be used as genetic markers to distinguish isolates collected from same region.