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Abstract Wheat blast disease caused by a South American lineage of Magnaporthe oryzae Triticum (MoT) pathotype has emerged as a serious threat to wheat production in Bangladesh since its first emergence in 2016. Efficient and suitable methods for isolation, storage, inoculum production and molecular characterization of the pathogen can help in achieving the target of sustainable management of the disease in a relatively short period of time. In this study, we aimed to develop suitable methods for isolation, storage and morphological characterization and molecular identification of MoT isolates collected from the blast-infected wheat fields in Bangladesh. This process included modification of existing protocols that were available for a related fungal pathogen M. oryzae or de novo method development and validation. We developed suitable methods for isolation of MoT from field-infected plant samples using modified monoconidial isolation technique and produced abundant conidia from a single mycelial plate for in vivo pathogenicity assay in a reproducible manner. Cultural and morphological characterization of the isolates revealed that all Bangladeshi MoT isolates are of a single clonal lineage with similar cultural and morphological characters. Molecular detection of isolates with M. oryzae-specific primers Pot1 and Pot2 and MoT-specific primers MoT3F and MoT3R produced bands with the expected size from all wheat-infecting isolates. We also successfully established a PCR-based detection system based on a commercially available detection kit for field-infected leaf and seed samples by detecting Pot2- and MoT3-specific bands. Additionally, the simple method we developed in our study for producing abundant conidia in a very short period of time will be very helpful in studying biology of the wheat blast fungus. This method was also proven to be more user-friendly and cost-effective than previously available methods. Successful characterization of MoT isolates at morphological and molecular levels coupled with detection of the pathogen in infected field and seed lots should be useful for efficient surveillance and management of the fearsome wheat blast disease.