Wheat (Triticum aestivum L.) is an allohexaploid (2n=6x=42; AABBDD) plant consisting of three subgenomes contributed by Triticum urartu (AA), Aegilops speltoides (BB), and Aegilops tauschii (DD). Due to its high nutrient value, wheat is known to be an important cereal crop and staple food worldwide. The consumption of wheat is expected to increase by 10% by 2050 in developing countries and prerequisite to increasing its production. Leaf rust, caused by Puccinia triticina, a biotrophic basidiomycetes fungal pathogen, has critically threatened wheat productivity and reduced the yield by approximately 10% annually. The urediniospores spread to almost all wheat-growing regions through wind dissemination and infect the leaves of wheat under favorable conditions. The practice of resistant cultivars was supposed to solve this problem, but the quick evolution of the pathogen overcame the resistance. Therefore, fetching solutions to leaf rust disease is a great challenge. F-box proteins are components of the SCF complex and have an essential role in substrate recognition and degradation accompanied by 26S proteasome. Very little studies are available for fungal F-box proteins belonging to basidiomycetes in contrast to ascomycetes fungi and plants. The present study includes genome-wide identification of F-box genes, their characterization using bioinformatics approaches, and their response to pathogenicity. A total of 10 F-box genes were unveiled in Puccinia genome sequences that had conserved F-box motifs at N-terminus along with leucine-rich repeat (LRR) and WD40 at C-terminus. The F-box proteins with similar domains clustered together in the phylogenetic tree while some clustered distantly, which might be due to functional diversity. GG705655 showed proximity with MoMet30, the pathogenic F-box gene in Magnaporthe oryzae. GG705409 clustered in a single clade with TaAFB4/TaAFB5 suggesting their possibilities of horizontal gene transfer during the coevolution of wheat and P. triticina. The F-box genes also showed orthologous relationship with other rust pathogens, Puccinia graminis, and Puccinia striiformis. These genes were interrupted by various numbers of introns and annotated to be components of SCF complex that participate in protein ubiquitination degradation process. The expression analysis of five F-box (GG705482, GG705421_1, GG705409, GG705553, GG711975) genes during during progressive stages of pathogenesis was performed. Among these, one gene GG705409 revealed positive response to pathogenicity at early stages of infection while GG711975 and GG705482 at later stages of infection. This study provides information on structure function elucidation of F-box proteins of a plant pathogenic fungi and their role during pathogenesis.