Background: Direct-acting antiviral agents (DAAs) for treatment of chronic HCV had proven an outstanding efficacy with low rates of virologic failures. However, the pre-existence and on-treatment selection of resistant subpopulations to the DAA in use can contribute to treatment failure. Objectives: To investigate the pattern of NS5A resistance associated substitutions (RASs) related to failure of Sofosbuvir/ Daclatasvir combination therapy in HCV genotype 4a infected Egyptian patients in comparison to baseline sequences from DAA-naïve patients, infected with HCV genotype 4a deposited in the GenBank. Methods: A 100 chronic HCV genotype 4a patients who achieved an end of treatment response to a 12 weeks course of Sofosbuvir/Daclatasvir combination therapy were tested for achieving sustained virologic response (SVR). In patients with relapse, characterization of RASs in NS5A domain I was done by direct sequencing technique after amplification by reverse transcriptase nested PCR. Baseline mutations prevalent in NS5A region of HCV genotype (GT) 4a were determined by studying similar sequences from DAA inexperienced GT4a patients deposited into the GenBank. Results: Seven out of 100 chronic HCV patients showed a relapse. 75% of patients experiencing a relapse showed NS5A RASs; 2 patients (50%) showed 2RASs and 1(25%) showed 3 RASs. Meanwhile, 17% of the studied GT4a sequences from the GenBank showed baseline NS5A RASs; 1sequence (2.86%) showed 2 baseline RASs and 5 (14.29%) show 1 baseline RAS. Conclusion: NS5A RASs were detected at higher rates in chronic GT4a patients with relapse. Their role in DAA treatment failure in presence of other negative predictors should be thoroughly investigated.