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Pakistan is facing major problem of viral based hepatitis which is increasing day by day, and is considered to be the major cause of mortality and morbidity. The present study investigated the frequency of Hepatitis B patients referred to Khyber Teaching Hospital (KTH) Peshawar from the different regions of Khyber Pakhtunkhwa. The blood sample was collected from the referred 1,33,193 patients and used for in vitroimmuno chromatographic assay (qualitative determination of HBsAg in serum or plasma), and ELISA (Enzyme Linked Immuno Sorbent Assay).Our results showed that 81(2.03%) out of 3,977 patients referred forHBsAg and anti-HCV screening presented positive test results for HBsAg. Similarly Co-infection with HCV was found in six patients (i.e. 4 females and two males). We conclude that transfusion of unscreened blood and blood products, use of contaminated syringes, lack of awareness about hepatitis and absence of hygienic conditions leads to its widespread occurrence.