Eucalyptus rust caused by Puccinia psidii is responsible for losses of approximately 20% of young Eucalyptus plants, depending on the environmental conditions and the geographic location. Despite its economic importance, there are few studies describing the genetic variability in P. psidii populations that infect different host plants. In the present study, we evaluated the ribosomal DNA internal transcribed spacer region (rDNA-ITS) using polymerase chain reaction denaturing gradient gel electrophoresis and terminal restriction fragment length polymorphism to assess the genetic variability in P. psidii populations infecting different Eucalyptus spp. and hybrids, as well as guava, jabuticaba, and syzygium. These culture-independent methods were efficient in differentiating populations based on the host species from which they were collected. In general, the results from both techniques showed that the populations collected from guava, jabuticaba, and syzygium were different from and had a greater level of diversity than the Eucalyptus rust populations. The sequencing of cloned rDNA-ITS fragments confirmed that the vast majority of the profiles generated were from P. psidii. This analysis also revealed interesting single-nucleotide polymorphisms. Therefore, these culture-independent methods are suitable for the rapid assessment of genetic variability within and between populations of this biotrophic fungus on a variety of host species and could be a tool to study the evolution of this pathogen and its interactions with host plants.