This investigation conducted a comprehensive evaluation of fire blight resistance across 258 Malus sieversii accessions representing seven natural populations in the Tianshan Mountains of Xinjiang, China. Utilizing standardized in vitro inoculation protocols for tender leaves, young shoots, and fruitlets with Erwinia amylovora strain E.aYZ003, complemented by longitudinal field surveillance of natural disease progression Results revealed distinct resistance patterns across tissues: fruitlets predominantly exhibited high resistance (HR), while tender leaves and shoots were mostly moderately susceptible (MS). Integrated analysis of in vitro and field data identified 28 resources with moderate resistance (MR) or higher. Among these, three accessions—‘GJS-4’, ‘GJS-12’, and ‘YA1-5’—demonstrated resistance (R) or higher across all tested tissues and field conditions. Significant inter-population variation was observed: the Gongliu population harbored the highest proportion of resistant resources, followed by Huocheng and Xinyuan populations. Yining County and Chabuchaer populations displayed intermediate resistance levels, whereas Emin and Tuoli populations exhibited the lowest resistance frequency. These study systematically characterized fire blight resistance in Malus sieversii germplasm, elucidated tissue-specific and population-level resistance variations, and identified promising resistant materials, providing a theoretical foundation for screening disease-resistant apple resources and fundamental materials for apple disease-resistant breeding programs.