Background: Anti-erythropoietin (EPO) antibodies against a recombinant form of Erythropoietin were associated with pure red cell aplasia. These antibodies probably cross-react with endogenous EPO causing severe anemia. Objective: This study was done to estimate the prevalence of anti-EPO antibody in prevalent hemodialysis patients and its relation to anemia and EPO resistance. Patients and Methods: This is cross-sectional study included 90 End-Stage Renal Disease (ESRD) patients who were on recombinant human erythropoietin for > 6 months with hemoglobin< 11 g/dL. Serum anti-EPO antibody was measured for all Patients that were divided into 2 groups: anti-EPO antibody positive and anti-EPO antibody negative. Results: The prevalence of anti-EPO antibodies was 45.6% (41 patients). 58.5% (24 patients) received EPO treatment by S.C route. Anti-EPO antibody-positive patients Showed lower hemoglobin level, serum iron, and Transferrin saturation with mean ±SD values 8.60±0.99 g/dl, 52.91±16.30 mcg/dl, 22.62±8.27 % respectively also lower MCHC with median (IQR)29.7 (2.50) g/dl. On comparing it with antibody-negative patients (N=49) there were significant differences with p values (< 0.0001), (0.038), (0.034), (0.002) respectively. These patients received higher EPO doses with high erythropoietin resistance index with median (IQR) values 8000 (4000) IU/week, 10.79 (8.495) respectively. The anti-EPO antibody was negatively correlated with hemoglobin  {r-0.661, p value 0.0001} and positively correlated with EPO doses, iron doses, and erythropoietin resistance index {r 0.309, p value 0.011, r 0.266, p value 0.003, r 0.417, p value 0.0001} respectively. Conclusions: It could be concluded that anti-EPO antibodies are commonly present in prevalent hemodialysis patients and were associated with resistant anemia and higher doses of EPO therapy.