Polymyxa species transmit viruses to many important crops. They are poorly understood obligate parasites occupying a distinct position in the Tree of Life. To better understand the potential for spread of Polymyxa-vectored diseases, ribosomal DNA was analysed from isolates covering a wide range of geographical locations, virus associations and hosts. Internal transcribed spacer 2 structure analysis indicated that Polymyxa graminis isolates could represent many species and there was more sequence variation within the known subgroups (ribotypes) than previously described. In cereal crops and soils from temperate climates Polymyxa isolates were usually ribotype I or II, but their host specificities or preferences were unclear. For the first time, there was evidence that ribotype I (in addition to ribotype II) could transmit SBWMV/SBCMV. Different ribotypes often occurred together in the same soil or plant. New hosts were identified for particular ribotypes, including the first detection of the sugar beet-infecting Polymyxa betae, in wheat. Unexpectedly, ribotype III-like sequences, usually restricted to crops in the tropics, were found in wheat from the USA. P. betae isolates showed limited variation (≤ 2%) and the recent change in susceptibility of sugar beet varieties to BNYVV in the USA is unlikely to be due to changes in P. betae.