Two DNA fragments, a 730-bp and a 900-bp fragment, one homologous to host cultivar specificity genes <i>nolBT</i> of <i>Sinorhizobium fredii</i> and the other one homologous to RSα, an insertion-like sequence present in <i>Bradyrhizobium japonicum</i>, were generated by polymerase chain reaction (PCR) with two pairs of primers. The amount of each fragment generated by the multiplex PCR was proportional to the amount of template DNA present. The amplification of the 900-bp RSα fragment was more sensitive, since it was amplified from a smaller amount of template DNA than the 730-bp <i>nolBT</i> fragment. By running the multiplex reaction in the presence of template DNA isolated from different sources, we confirmed that the reaction can discriminate between <i>S. fredii</i>, <i>Bradyrhizobium japonicum</i> and <i>Sinorhizobium xinjiangensis</i>.Facultad de Ciencias Agrarias y ForestalesInstituto de Fisiología Vegetal