Polymyxa graminis f. sp. temperata and P. graminis f. sp. tepida are distinguished on the basis of their specific ribosomal DNA sequences. In order to evaluate whether or not host specialization is associated with the special form, the occurrence of infection of both forms on barley and wheat was studied. P. graminis inocula were obtained from soils collected in Belgium and France. Their ribotypes were characterized using molecular tools specific to P. graminis f. sp. temperata or P. graminis f. sp. tepida such as restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR)-amplified rDNA, nested and multiplex PCR. Both special forms were found in each country and coexisted in some soils. The host specificity of P. graminis special forms for barley and wheat was studied from two soils collected at Gembloux (Belgium) and Chambon-sur-Cisse (France), each infested by bymo- and furoviruses. P. graminis f. sp. temperata is more frequent on barley and P. graminis f. sp. tepida on wheat. Furthermore, the quantification of each form on barley and wheat by two separated real-time quantitative PCR assays confirms the observations on the vector specialization. These results suggest a certain but not exclusive host specificity of P. graminis special forms.