OBJECTIVE About 248 million people are chronic HBV surface antigen carriers in the world. Hepatitis D virus (HDV) infection present in more than 15 million people worldwide. HDV needs hepatitis B surface antigen (HBsAg) to help its replication. We aimed to estimate the prevalence of HDV infection among HBsAg positive individuals and to determine the clinical, laboratory and virological characters of HDV infected patients. METHODS This study was prospective cross-sectional analytic one including 186 HBsAg positive cases. Anti-HBc total, IgM and HBV PCR were done for all of these cases. Anti-HDV ELISA analysis was done for all cases. Positive samples for Anti-HDV by ELISA were then tested by HDV PCR. RESULTS Of the 186 HBsAg positive cases, 80 were reactive for anti-HDV antibodies, resulting in an overall anti-HDV seropositivity of 43%. Higher prevalence of liver cirrhosis (43.8%), HCC on top of cirrhosis (8.8%) were found in anti-HDV positive compared to anti-HDV negative cases (17.9%) and (3.8%) respectively (p value<0.001). Portal hypertension and Child-Pugh grade B, C were significantly higher in anti-HDV-positive cases as compared to the anti-HDV-negative ones (47.5% versus 18.9%) and (11.3% versus 6.6%); (16.3% versus 3.8%) respectively (p value<0.001 for each). HDV RNA was positive in 25 out of 80 anti-HDV-positive cases (31.3%). CONCLUSIONS Anti-HDV was seropositive in 43% among HBsAg positive cases in Upper Egypt. HDV RNA was positive by PCR in 25 out of 80 anti-HDV-positive cases (31.3%). HDV prevalence using PCR was 25/186 (13.4%) in Upper Egypt.