The practical use of molecular markers is facilitated by cost-effective detection techniques. In this work, wheat insertion site-based polymorphisms (ISBP) markers were set up for genotyping using high-resolution melting analysis (HRM). Polymorphic HRM-ISBP assays were developed for wheat chromosomes 4A and 3B and used for wheat variability assessment. The marker sequences were mapped against the wheat genome reference sequence, targeting interesting genes. Those genes were located within or in proximity to previously described quantitative trait loci (QTL) or meta-quantitative trait loci (MQTL) for drought and heat stress tolerance, and also yield and yield related traits. Eighteen of the markers used tagged drought related genes and, interestingly, eight of the genes were differentially expressed under different abiotic stress conditions. These results confirmed HRM as a cost-effective and efficient tool for wheat breeding programs.