Switchgrass rust caused by Puccinia novopanici (P. novopanici) has the ability to significantly affect the biomass yield of switchgrass, an important biofuel crop in the United States. A comparative genome analysis of P. novopanici with rust pathogen genomes infecting monocot cereal crops wheat, barley, oats, maize and sorghum revealed the presence of larger structural variations contributing to their genome sizes. A comparative alignment of the rust pathogen genomes resulted in the identification of collinear and syntenic relationships between P. novopanici and P. sorghi; P. graminis tritici 21–0 (Pgt 21) and P. graminis tritici Ug99 (Pgt Ug99) and between Pgt 21 and P. triticina (Pt). Repeat element analysis indicated a strong presence of retro elements among different Puccinia genomes, contributing to the genome size variation between ~1 and 3%. A comparative look at the enriched protein families of Puccinia spp. revealed a predominant role of restriction of telomere capping proteins (RTC), disulfide isomerases, polysaccharide deacetylases, glycoside hydrolases, superoxide dismutases and multi-copper oxidases (MCOs). All the proteomes of Puccinia spp. share in common a repertoire of 75 secretory and 24 effector proteins, including glycoside hydrolases cellobiohydrolases, peptidyl-propyl isomerases, polysaccharide deacetylases and protein disulfide-isomerases, that remain central to their pathogenicity. Comparison of the predicted effector proteins from Puccinia spp. genomes to the validated proteins from the Pathogen–Host Interactions database (PHI-base) resulted in the identification of validated effector proteins PgtSR1 (PGTG_09586) from P. graminis and Mlp124478 from Melampsora laricis across all the rust pathogen genomes.