[Objectives]  To explore the infection of jujube witches’-broom (JWB) phytoplasma and Wolbachia in different populations of the leafhopper, Hishimonus lamellatus Cai et Kuoh and the distribution of Wolbachia in different organs and tissues of the leafhopper, and to clarify the infection types and taxonomic status of Wolbachia in Hishimonus at jujube orchard, which laid a foundation for the study on the effects of Wolbachia infection on the biology and ecology of potential vectors of jujube witches’-broom phytoplasma. [Methods]  Molecular detection and identification of field and laboratory populations of Hishimonus lamellatus were carried out by using gene-specific primers of Phytoplasma and Wolbachia. [Results]  The infection rate of JWB-phytoplasma of natural populations of Hishimonus lamellatus adults collected in jujube orchards was 55%-61%, while the infection rate of Wolbachia was 3%-4%. When the natural populations of the leafhopper are reared and laid eggs，which hatched to nymphs，Wolbachia was detected in the 1st to 4th instar nymphs，and phytoplasma was detected in the 2nd to 5th instar nymphs. Wolbachia infection was found in ovaries, eggs and nymphs of the laboratory colonies reared the leafhopper phytoplasm-free, and was also detected in salivary glands and alimentary tract, with infection rates was between 58% and 100%. Phylogenetic tree was constructed based on wsp gene of Wolbachia. It was found that two Wolbachia strains belonging to group B belonged to novel strains wLam1 and wLam2, which were different from other strains of group B. [Conclusion]  The natural populations of adult leafhoppers could be infected with JWB-phytoplasma and Wolbachia. The colonies of adult leafhoppers phytoplasma-free reared in the laboratory were infected with Wolbachia significantly more than those from the natural populations. Two Wolbachia strains in Hishimonus belonged to group B. The results provided basic information for the further use of Wolbachia as a biocontrol agent for leafhopper vectors.