Pennisetum species have importance in foraging, agriculture, energy-production, the environment, and landscaping. To promote the preservation and utilization of ornamental Pennisetum resources, we developed simple sequence repeat (SSR) markers from the Pennisetum setaceum cv. ‘Rubrum’ transcriptome and verified their conservation in 38 sources. Our transcriptome sequencing efforts generated 58.91 Gb of clean data containing 55,627 unigenes. We functionally annotated 30,930 unigenes, with functions enriched in translation and ribosomal structure and biogenesis. Database comparisons indicated that the closest relative of P. setaceum cv. ‘Rubrum’ is Setaria italica. Over five thousand SSR markers were detected in the transcriptomic data. We selected 38 pairs of highly polymorphic SSR markers from 50 randomly selected SSR markers. Based on genetic diversity analysis of 38 ornamental Pennisetum sources, we obtained 312 polymorphic bands, with an average of 8.21 alleles per primer. Principal coordinate analyses and generation of a, which proved that Pennisetum has moderate genetic diversity. In addition, fingerprint maps were constructed to improve Pennisetum identification. The transcriptome data generated by our study enhances the transcriptional information available for P. setaceum. This study lays the foundation for the collection and utilization of ornamental Pennisetum resources and provides a basis for future breeding projects using this species.