A novel phytoplasma was detected in a cherry plum (Prunus cerasifera Ehrh) tree that mainly showed yellow leaf symptom. The tree was growing in an orchard located in Yangling District, Shaanxi Province, China. The leaves started as chlorotic and yellowing along leaf minor veins and leaf tips. Chlorosis rapidly developed to inter-veinal areas with the whole leaf becoming pale yellow in about 1-4 wk. Large numbers of phytoplasma-like bodies (PLBs) were seen under transmission electron microscopy. The majority of the PLBs was spherical or elliptical vesicles, with diameters in range of 0.1-0.6 μm, and distributed in the phloem cells of the infected tissues. A 1246-bp 16S ribosomal RNA (rRNA) gene fragment was amplified from DNA samples extracted from the yellow leaf tissues using two phytoplasma universal primer pairs R16mF2/R16mR1 and R16F2n/R16R2. Phylogenetic analysis using the 16S rRNA gene sequence suggested that the phytoplasma associated with the yellow leaf symptoms belongs to a novel subclade in the aster yellows (AY) group (16SrI group). Virtual and actual restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene fragment revealed that the phytoplasma was distinguishable from all existing 19 subgroups in the AY group (16SrI) by four restriction sites, Hinf I, Mse I, Sau3A I and Taq I. The similarity coefficients of comparing the RFLP pattern of the 16S rRNA gene fragment of this phytoplasma to each of the 19 reported subgroups ranged from 0.73 to 0.87, which indicates the phytoplasma associated with the cherry plum yellow leaf (CPYL) symptoms is probably a distinct and novel subgroup lineage in the AY group (16SrI). In addition, the novel phytoplasma was experimentally transmitted to periwinkle (Catharanthus roseus) plants from the tree with CPYL symptoms and then back to a healthy 1-yr-old cherry plum tree via dodder (Cuscuta odorata) connections.