Patients with thalassemia major are at high risk of hepatitis C through blood transfusion from donors infected by hepatitis C virus (HCV). The use of direct-acting antiviral (DAA) therapy against such HCV infections has increased in different populations. However, resistant viral variants can affect treatment outcomes, and therefore improved surveillance strategies are needed. Accordingly, we aimed to evaluate resistance-associated substitutions (RASs) to HCV DAAs at the baseline of treatment in thalassemia patients in a referral center. Out of 89 thalassemia patients who suffered from HCV infection and were referred to our center between 2016 and 2017, 43 underwent further analysis of the HCV nonstructural proteins NS5A and NS5B using polymerase chain reaction (PCR) sequencing methods. Unique primers were designed using bioinformatics software for separate detection of HCV subtypes 1a, 3a, and 1b. Detection of RASs was performed based on previously published literature. Statistical analysis was carried out using SPSS version 19. The participants, 60.4% (26/43) of whom were male, had a mean age ± standard deviation (SD) of 33.0 ± 5.0 years. HCV subtype 1a was found in 27 cases, 3a in 13, and 1b in three. In HCV subtype 1a there were 163 mutations in NS5A and 212 mutations in NS5B. The frequency of RASs was 20.9% (8 RASs in 9 patients), including M28V and H58P in subtype 1a, L28M, R30Q, C316N, and C316S in subtype 1b, and S24F in subtype 3a. Statistically, the subtype 1b and a higher mutation rate in NS5A were associated with RASs (p-value < 0.05). The emergence of natural RASs to HCV DAAs serves as a warning of the risk of drug resistance in response to the broad usage of antivirals. However, relapses in these DAA-treated HCV-infected thalassemia patients are rarely reported. Our findings indicate that the prevalence of RASs prevalence at baseline was 20.9% in these patients, and this calls for extrapolation to a larger population study, as highlighted in other studies, with larger sample sizes, high-throughput methods, and follow-up in order to fully evaluate treatment outcomes in RASs-detected individuals. Optimized therapeutic strategies, particularly in complex, difficult-to-cure patients, can effectively prevent DAA treatment failure as a result of selection for RASs.