Proteins participate in defense mechanisms after a pathogen attacks a host. Pathogenesis-related (PR) proteins in wheat plants play the important role of defense against Puccinia striiformis f. sp. Tritici. This study uses various analytic methods to describe the expression of five (PR) protein genes (PR1, PR2, PR4, PR9 and PR10) and genetic polymorphism among sixteen wheat genotypes to understand the mechanisms underlying of PR genes in Egyptian wheat compared with the Yr genotype. The PR protein genes were semi-quantitatively and quantitatively assessed in four selected wheat genotypes; two resistant (Misr-3 and Yr-29) and two susceptible (Sids-12 and Avocet-S). Reverse transcriptase PCR analysis results indicated that all PR protein genes exhibited different expression levels among the four studied genotypes and overexpression at the resistant genotypes in the different time points of Pst inoculation. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that the expression of PR protein genes was elevated in adult plant resistant (APR) genotypes. Plant stage in combination with the infection process by stripe rust provided even greater upregulation in the expression of PR genes and enhanced induction of defense enzyme activities in the same sample at different time points for various wheat genotypes. •Sixteen wheat genotypes were evaluated under artificial infection with stripe rust, Misr-3 was the most resistant genotype.•Five PR protein genes were used to evaluate and produce unique DNA profiles for the studied genotypes.•Expression of PR protein genes was more effective in adult plant than the seedling.•Induction of PR protein genes was correlated with CAT and POX defense enzyme activities at the same time point.