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<p>Abstract</p> <p>Background</p> <p>Hepatitis delta virus (HDV) and Hepatitis B virus (HBV) co-infection is well known to induce a spectrum of acute and chronic liver diseases which further advance to cirrhosis, fulminant hepatitis and hepatocellular carcinoma (HCC).</p> <p>Aim</p> <p>The aim of the present study was to determine the prevalence of hepatitis D virus super-infection among hepatitis B surface antigen (HBsAg) positive individuals in the highly populated province of Pakistan which is not well known.</p> <p>Methods</p> <p>Sera samples were subjected to HBsAg and anti-HDV screening and finally anti-HDV and HBsAg positive coinfected samples were used for HDV active RNA confirmation using nested polymerase chain reaction (PCR).</p> <p>Results</p> <p>Out of total 200 HBsAg positive samples by rapid device, 96 (48%) were also found reactive for HBsAg using enzyme linked immunosorbant assay (ELISA). Out of these HBsAg ELISA positive samples, 80 (88.8%) were anti-HDV ELISA positive which were then subjected to PCR. The amplification results further confirmed 24 (30%) samples to be HDV RNA positive. HDV super-infection was more common in male patients than female patients (81% VS 19%).</p> <p>Conclusion</p> <p>The current study shows a high prevalence rate of HDV-HBV co-infection in Pakistan that tends to increase over time.</p>