Brown rot or bacterial wilt of potato caused by Ralstonia solanacearum ( Yabuuchi et al. 1995) is one of the most quarantine disease world wide as well as in Egypt. Therefore, isolation and identification of the causal organism from different sources using rapid and accurate methods was carried out. Results revealed that, from 200 collected samples, 20 pathogenic isolates were detected from potato tubers, weeds, water and soil. Fifteen isolates were isolated from Mounfia governorate, five from El- Gharbia and non from El–Sharkia (Salhia free area). Pathogenic capability of these isolates were confirmed on tomato seedlings. Soil isolates (S1 and S2), tuber isolate (No. t8) and water isolates (No. w12) showed the highest disease severity of tomato seedlings. The aforementioned twenty pathogenic isolates were identified as Ralstonia solanacearum Race 3, Biotype 2 according to their biochemical, and physiological properties, inoculation on King's B and SMSA media, Immunofluorecence antibody assay (IFAS), Fatty acid assay, DNA assay using PCR and Box-PCR. Ralstonia solanacearum the causal organism of potato brown rot could be stored for three months at pH 7 and 15°C.