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Barley leaf stripe, caused by Pyrenophora graminea, is an essential systemic seed-borne disease in barley worldwide. Barley is a major cereal crop in the Qinghai–Tibet Plateau, and barley production has been threatened by leaf stripe in this region, particularly in organic farming regions. Detecting the pathogen in infected barley seeds is crucial for managing barley leaf stripe. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed to detect the pathogen based on primers designed based on the sequence of the pig 14 gene (GenBank: AJ277800) of P. graminea. The optimal concentrations of MgSO4, dNTPs, and enzymes in the LAMP reaction system were established as 10.0 mM, 1.0 mM, and 8 U in a 25 μL reaction volume, respectively. The established LAMP methods for detecting P. graminea were optimally performed at 63 °C for 70 min with high reliability. The minimum detection limit was 1 × 10−2 ng·μL−1 in the 25 μL reaction system. The specificity of LAMP for P. graminea was validated with eight fungal species. All DNA extracts from P. graminea-infected barley seeds with incubation, intact, and smashed treatments were applied in LAMP and confirmed to enable the detection of the pathogen. The LAMP assay in this study could facilitate the detection of P. graminea in barley seeds onsite, provide information for seed health certificates, and help decide on seed treatment in leaf stripe management. [ABSTRACT FROM AUTHOR]