Hepatitis delta virus (HDV) infection results in more severe and even fulminant form of hepatitis B in co-infected cases. This study was designed to estimate the prevalence of anti-HDV positivity and the associated risk factors in patients with chronic hepatitis B virus infection in Zahedan (Iran). In this cross-sectional study a total of 440 consecutive patients with chronic hepatitis B virus (HBV) infection attending the Zahedan Gastroenterology and Hepatology clinics from 2008 to 2011 were included. We performed test for HDV serum marker, using commercially available enzyme-linked immunosorbent assay kit. Patients were split into two groups according to their HDV antibody status as HDV positive or negative. The collected data were coded, and the statistical analyses were conducted. Four hundred and forty patients with various forms of chronic HBV-related liver diseases enrolled in the study. 200 (45.5%) patients were carrier for HBV. 196 (44.5%) patients had chronic active hepatitis and 44 (10%) patients suffered from cirrhosis. Anti-HDV was demonstrated in 75 patients (17%). The prevalence of HDV was 7%, 16.3% and 65.9% in carriers, patients with chronic active hepatitis and cirrhosis, respectively. HDV infection is still an important public health problem in Zahedan and appears a major cause of progression of liver disease induced by HBV.