To further understand the genomic diversity and genetic architecture of phytoplasmas, a physical and genetic map of the sweet potato little leaf (SPLL) strain V4 phytoplasma chromosome was determined. PFGE was used to determine the size of the SPLL-V4 genome, which was estimated to be 622 kb. A physical map was prepared by two-dimensional reciprocal digestions using the restriction endonucleases BssHII, Smal, Eagl and I-Ceul. Sixteen cleavage sites were located on the map. Southern hybridizations of digested SPLL-V4 chromosomal DNA were done using random clones and PCR-amplified genes as probes. This confirmed fragment positions and located the two rRNA operons and the linked fus/tuf genes encoding elongation factors G and Tu, respectively, on the physical map. An inversion of one of the rRNA operons was observed from hybridization data. Sequence analysis of one of the random clones identified a gid gene encoding a glucose-inhibited division protein. Digestions of the tomato big bud (TBB) phytoplasma chromosome with the same four enzymes revealed genome heterogeneity when compared to the closely related SPLL-V4, and a preliminary chromosome size for the TBB phytoplasma of 662 kb was estimated. This mapping information has revealed that significant genome diversity exists within the phytoplasmas.