Phytoplasmas in the aster yellows (16SrI), clover proliferation (16SrVI) and stolbur (16SrXII) groups have a wide host range with common hosts. In the current study, specific primers based on the 16-23S rRNA spacer region were designed and optimised for the detection and identification of phytoplasmas in these groups in a multiplex nested PCR assay. Fifty-two phytoplasma symptomatic potato plants were tested, and clover proliferation (16SrVI) and stolbur (16SrXII) phytoplasmas detected coinfecting 16 potato plants with single infection of these phytoplasmas in 5 and 21 samples, respectively. ‘Candidatus P. asteris’ (16SrI) was not detected. The specificity of the primers was proven in both single and multiplex PCR assays. Detection of multiple infections can be useful in assisting epidemiological studies and in deciding appropriate disease management strategies.