Spiroplasma citri, the causal agent of citrus stubborn disease, is widely distributed in citrus groves and causes heavy losses to the citrus industry in southern Iran. In the present study the genetic diversity of S. citri strains isolated from various hosts and locations in the Fars Province in southern Iran was investigated. The bacterium was cultured from stubborn affected citrus trees, sesame and safflower plants, and leafhopper vector Circulifer haematoceps. Based on PCR-RFLP of the spiralin gene (spi), four groups could be clearly identified among Iranian S. citri isolates. Group 1 was prominent with 71.8% of isolates found in both citrus and non-citrus hosts. Members of groups 2 (18%), and 5 (5.1%) were identified only in citrus, and members of group 6 only in non-citrus hosts. Phylogenetic analyses based on spi gene sequence could not clearly discriminate isolates of groups 1 and 2. In most cases, RAPD-PCR allowed the differentiation of isolates in different clades based on location or host. However there was no correlation between classification based on RFLP of spi gene and RAPD-PCR of whole genome. Finally, the study was completed by the demonstration that Iranian isolates found only in non-citrus hosts (group 6) including insect vectors are capable of infecting key lime when experimentally transmitted to the plant via a dodder bridge.