Puccinia striiformis f. sp. tritici (Pst), the causative agent of wheat stripe rust, poses a significant threat to wheat production due to its rapid long-distance migration and epidemic properties. Understanding the genetic structure and dynamics of the Pst population is crucial for early prediction and establishment of effective control strategies. The types of molecular marker analyses used in previous population genetic studies are often costly, time-consuming, and labor-intensive. We developed a genotyping by target sequencing (GBTS) chip for Pst designed with candidate secretion proteins and highly polymorphic single nucleotide polymorphism (SNP) sites identified from genome resequencing. The chip can be used directly with diseased leaves, saving time and avoiding cross-contamination between samples. The feasibility and efficiency of the chip was tested using 225 infected leaf samples collected from the northwest oversummering region of China. This test yielded 1,293,150 high-quality SNPs with a maximum gap of 99,512 bp. Strict quality controls produced 19,139 SNPs, comprising the final Pst 20K GBTS chip. Population genetic analysis revealed frequent gene flow and similar genetic diversity of Pst between epidemic regions, consistent with wind field analysis, trajectory tracking, and field monitoring. The results demonstrated that the GBTS chip is more efficient, convenient, and lower in cost than previous methods. This study provides new insights into stripe rust population dynamics. Furthermore, the newly established chip offers a valuable method for enriching epidemiological recognition, guiding future research into inter-regional or continental transmission of an important plant pathogen.