Fungicide abuse leads to the emergence of fungicide-resistant fungal pathogens, thus posing a threat to agriculture and food safety. Here, we developed an isothermal amplification refractory mutation system (termed iARMS) allowing us to resolve genetic mutations, enabling rapid, sensitive, and potentially field-applicable detection of fungicide-resistant crop fungal pathogens. iARMS yielded a limit of detection of 25 aM via a cascade signal amplification strategy of recombinase polymerase amplification (RPA) and Cas12a-mediated collateral cleavage at 37 °C within 40 min. Specificity for fungicide-resistant Puccinia striiformis (P. striiformis) detection was guaranteed by RPA primers and the flexible sequence of gRNA. The iARMS assay allowed us to detect as low as 0.1% cyp51-mutated P. striiformis that showed resistance to the demethylase inhibitor (DMI), which was 50 times more sensitive than the sequencing techniques. Thus, it is promising for the discovery of rare fungicide-resistant isolates. We applied iARMS to investigate the emergence of fungicide-resistant P. striiformis in western China and found that its proportion was over 50% in Qinghai, Sichuan, and Xinjiang Province. iARMS can serve as a molecular diagnostic tool for crop diseases and facilitate precision plant disease management.