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Fungal diseases, especially leaf and stripe rusts are wheat yield reducing factors in Iran and the world. In this study, genetic diversity of 20 wheat varieties with different response to leaf and stripe rusts were studied using primers designed based on the conserved regions of plant disease resistance genes. The banding patterns of polymorphic markers were scored as dominant and number of amplified bands and percentage of polymorphism were determined. In addition, for each primer combination, polymorphic information content (PIC) and marker index (MI) were calculated. Out of the 11 used single primers and primer combinations, five primer combinations and a single primer produced scorable amplification. The maximum and minimum of PIC were observed for LLOOP-1 and H2016-H2020 primer combination with mean value of 0.50 and 0.28, respectively. The primer combinations of H2016-H1146 and H2016-H2020 with mean values of 4.80 and of 2.84, had minimum and maximum of MI, respectively. Cluster analysis based on Neighbor-Joining algorithm and evolutionary P-distance coefficient assigned the varieties into four groups which were in agreement with their response to yellow rust. In principal coordinate analysis, the scatter plot of varieties based on two first coordinates confirmed the groups obtained from cluster analysis.