Citrus fruit is considering very nutritious fruit. Citrus trees are affected by the several viruses, bacteria, fungi and virus-like diseases. In latest years, citrus trees are seriously affected by a prokaryotic pathogen Spiroplasma citri that cause stubborn disease. On affected trees, fruits become small, crooked and seeds are aborted. The present study was planned to diagnose the pathogen through Sp4 media. Spiroplasma citri was isolated from infected leaves and columella part of fruit. Culture inoculated cultured media were incubated at 300C. After 7-14 days of incubated media color was changed from red to yellow which showed the initial presence of S. citri. Typically, fried egg shape colony was observed on solid media. Molecular detection of Spiroplasma citri, DNA was extracted from leaves sample. Spiroplasma specific Spiraline-f / and spiraline-r primer pair was used in to amplify spiraline gene with 675bp product. Pathogenicity test was performed. Upon successful grafting, symptoms were observed that confirmed the presence of disease. Moreover, detection of citrus stubborn leads to the first-time identification of S. citri on media, biologically and molecularly in the citrus orchards of Sargodha.