Spiroplasma citri, the causal agent of stubborn disease, was isolated from leaves and fruits of diseased citrus plants and cultured on solid and liquid C3G medium. On the basis of mode of transmission, symptomatology, shape on solid medium, staining of infected plant tissues with Dienes’ stain and phase contrast microscopy, the isolated agent was identified as Spiroplasma citri. Identifiction was ensured by ELISA and PCR techniques. A fragment (1053 bp) from the spiralin gene of S. citri was amplified by PCR using two specific primers for the spiralin gene. The nucleic acid hybridization techniques (Southern and dot blots) were used for identification of the spiroplasmal genome using non-radioactive DNA probe specific for spiralin gene region. It was carried out as an alternative sensitive method for rapid detection of the Egyption isolate of S. citri   using non-radioactive DNA probe specific for spiralin gene region.