Background: PCR-coupled single-strand conformation polymorphism (PCR-SSCP) is often used as a first line screening technique to facilitate mutation detection in large genes such as the DNAH5 gene. A modified protocol of PCR-SSCP by primers addition before denaturation may improve banding profiles. Objectives: The aim is to study the effect of primers addition and determine the optimal conditions to screen DNAH5 hotspot exons. Materials and Methods: Prior to SSCP screening, various concentrations of primers were added to PCR-products of DNAH5 hotspot exons. The resolution and intensity of the electrophoretic bands were then evaluated. Results: Herein we prove that specific primers addition at 4 μM concentration improves the resolution and efficiency of the method without altering the detection of the mutations. Moreover, an optimal result was obtained by adding both primers for some exons and only the reverse one for others. Conclusion: The modified PCR-SSCP protocol described in this present study could be used to screen the mutations in DNAH5 hotspot exons in patients with primary ciliary dyskinesia (PCD).