Panicum virgatum L. is an important biomass energy plant. Its straw is rich in lignocellulose which can be used as raw material for ethanol production. An investigation on chromosome localization and distribution of 45S rDNA in P. virgatum cultivars with different ploidy would promote the cytogenetic study of the species. In this investigation, chromosome localization of 45S rDNA and karyotype analysis were conducted by fluorescence in situ hybridization using root tips of three tetraploids and six octaploids as materials. The results indicated that there were 36 chromosomes in the somatic nucleus of the tetraploids, which could be divided into nine groups, each with four homologous chromosomes. The karyotype formula of the tetraploids was 2n = 4x = 36 = 32m (SAT) + 4sm, and 45S rDNA was invariably located at one end of chromosome 3. Significant differences in number of chromosomes and location of 45S rDNA signals were found among different octaploid cultivars or different individuals of the same octaploid cultivar. Four types of 45S rDNA distribution in the octaploid cultivars existed. The first type had a strong 45S rDNA signal at the end of both arms of the chromosome. The second type had a strong 45S rDNA signal within one arm of the chromosome. The third type had a strong 45S rDNA signal at the end of one arm of the chromosome. The fourth type had a weak 45S rDNA signal within one arm of the chromosome. The complexity of the models of 45S rDNA signal distribution among different octaploid cultivars or different individuals of the same octaploid cultivar is inferred to be closely correlated to homologous recombination and structure variation of the chromosoms.