Technical Abstract: Spiroplasma citri, a phloem-limited, leafhopper-transmitted pathogen, causes citrus stubborn disease (CSD). Losses due to CSD in California orchards has grown over the past decade. To investigate the possibility of introduction or emergence of a new spiroplasma strain, a study of genetic diversity among S. citri strains from various locations was conducted using RAPD-PCR of 35 strains cultured from 1980-1993, and of another 35 strains cultured from 2005-2006. Based on analysis with 20 primer pairs, considerable diversity was observed among strains, however, no unique genetic signatures were associated with recently collected strains compared with those collected 15-28 years ago. Further, no geographically associated pattern was distinguishable. S. citri from carrot and daikon radish was found to contain some unique DNA fragments, suggesting some host plant influence. Multiple strains from single trees also showed genetic diversity. Sequencing of five RAPD bands that differed among S. citri strains showed that diversity-related gene sequences include virus fragments, a membrane lipoprotein, a DNA modification enzyme, and a mobilization element. No differences in colony morphology were observed among the strains. The lack of correlation between PCR patterns and isolation date or collection site is inconsistent with the hypothesis that recent infections are due to the introduction or emergence of novel pathogen strains.