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A monoclonal antibody was produced by using enriched antigen from an infected chokecherry plant, and was used to detect the X-disease phytoplasma using both ELISA and immunofluorescence staining. The monoclonal antibody reacted with five of seven tested phytoplasmas in the X-disease phytoplasma cluster, and one of four phytoplasmas (pigeon pea witches'-broom phytoplasma) outside that cluster. The efficiency of the monoclonal antibody for detection of X-disease phytoplasmas was verified through assays of infected stone fruit plants. The efficacy of the monoclonal antibody was confirmed by X-disease phytoplasma-specific nested-polymerase chain reaction. Development of the monoclonal antibody represents a substantial improvement in the potential for commercial diagnosis of X-disease phytoplasma infection in nurseries and orchards and for screening large numbers of stone fruit plants for X-disease phytoplasma infection in breeding programs.