Introduction: Hereditary spherocytosis is a heterogeneous disorder with mild to moderate anemia. The aim of this study was to evaluate the inherited spherocytosis gene mutations in patients with RBC cytoplasmic disorders in Iranian population. Materials and Methods: In this study, Whole Exome Sequencing (WES) was performed for patients suspected to hereditary spherocytosis and their relatives. Results: Sequence analysis of the probands and their parents identified variations in ANK1 gene (NM_001142446.1:c.127-2A>G), SPTB (c. 14delC, p.Thr5LysfsTer41), SPTA1 (c.466C>T), SLC4A1 (c.2494C>T) and SLC25A38 gene (c.683G>T, NP_060345.2:p.Gly228Val that could be related to the patients clinical manifestation. Conclusion: Findings are in line with the appropriate diagnostic yield of WES in determining the causative variant especially in those disorders that many genes are involved like anemia. This is the first report of a cohort of Iranian patients with anemia suspected to that were investigated using WES technology. Further studies are needed to investigate the distribution of gene mutations in patients with RBC membrane disorders in Iran