Structural variations (SVs) pervade plant genomes and contribute substantially to the phenotypic diversity of species. However, most SVs were ineffectively assayed because of their complex nature and the limitations of sequencing technologies. By sequencing wheat genomes using the PacBio high-fidelity (HiFi) sequencing technology, capable of generating long and highly accurate reads, we conducted a comprehensive evaluation of mainstream long-read aligners and SV callers for SV detection. The results showed the accuracy of SV discovery depends more on aligners than on callers. For aligners, pbmm2 and NGMLR provided the most accurate results while detecting deletion and insertion, respectively. For SV callers, cuteSV and SVIM achieved the best performance. We demonstrated that combining the aligners and callers mentioned above is optimal for SV detection. Furthermore, we evaluated the effect of sequencing depth on the accuracy of SV detection, showing that low-coverage HiFi sequencing is well qualified for high-quality SV discovery. These results will help advance SV discovery and unveil its biological functions in wheat and many other plants.