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Wheat blast, caused by Pyricularia oryzae pathotype Triticum, is one of the most notorious diseases of wheat. In this study, a total of twenty-four monoconidial isolates representing four major wheat blast affected districts, namely Chuadanga, Meherpur, Kustia and Jhenaidah of Bangladesh were analyzed. Eight RAPD and four ISSR primers being used for genetic diversity assay produced a total of 94 bands of which 85% were polymorphic. UPGMA dendrogram based on combined dataset (RAPD and ISSR) separated all the isolates into two main clusters having similarity ranged from 64 to 93%. Principal coordinate analysis showed congruent result with cluster analysis. However, clustering of the isolates according to their origin and plant part sampled was not apparent in the dendrogram. The genetic diversity indices unveiled that genetic diversity in P. oryzae populations is low. Average Nei's gene diversity (h) and Shannon's Information Index (I) calculated for isolates from each district were found 0.16 and 0.24, respectively. The population structure analysis of the isolates revealed the presence of two sub-populations with admixture of alleles. Analysis of molecular variance indicated that significantly higher level of variation (96%) in the population was present within districts while a relatively low proportion (4%) of the variation was detected among districts. Knowledge generated in this study will give a pace in the development of appropriate wheat blast management strategies to control this disease in Bangladesh., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)