NGS-based multiplex assay has accomplished a valuable status as a means of high throughput research, rapid screening functional markers in wheat breeding programs. Accordingly, we applied a total of 42 locus-specific markers from Indel and SNP-mediated categorisations coupled with the agronomic important genes or quantitative trait loci (QTL) in bread wheat. The amplicons were analysed by an Ion Torrent Proton Sequencer. Then, an allele detection custom pipeline was applied to process the genotype of a total of 99 Iranian cultivars and 270 landraces. On the whole, 29 markers were positively incorporated and achieved 100% SNP call rates. Assessment of sequence-tagged site (STS) and competitive allele-specific PCR (KASP) markers concerning the same loci confirmed the genotype calls of all markers altogether. It was revealed that the Iranian cultivars and landraces supply a rich genetic resource capable of resisting Hessian fly, leaf rust, fusarium head blight, adult plant leaf diseases, stem rust, wheat soilborne mosaic virus, wheat streak mosaic, pre-harvest sprouting, high grain protein, and gluten strength traits. This finding can be developed to improve and enrich bread wheat. Further, it is advocated that NGS-based multiplex assay can be a promising approach for high throughput in examining trait-linked markers in wheat germplasm collections.