Abstract : This retrospective cohort study aims to estimate the prevalence rate of Hepatitis B infected patients at the District Rawalpindi and Islamabad, Pakistan. A total of 490 infected patients’ complete medical records were collected with the help of a standard prepared questionnaire containing demographic details, hepatic complaints, past and present illness, physical examination records and other diagnostic laboratory reports. All 490 collected medical records were analyzed using GraphPad Prism (Version 8.40). The Pearson chi-square test was used with confidence level = (95 %) and a statistically significant P-value <0.05 was considered. Of 490 medical records, 72 (14.69%) patients found positive for Hepatitis B infection, including male 39 (18.93%) and female 33 (11.61%). Patients were more prevalent 3 (25%) in the age of <10 years. The highest prevalence was reported in the month of November 9 (32.14%) followed by February 18 (24%). Similarly, the higher ratio of 20 (19.41%) of Hepatitis B was reported in 2020 as compared to the 2021 year 52 (13.43%). Moreover, the highest seropositivity of HBsAg has resulted via quantitative Real-Time PCR 29 (27.88%), followed by Enzyme-Linked Immunosorbent Assay 39 (5.66%), qualitative PCR were 03 (10%) and by Immune-chromatography test was only 1 (0.93%) specimen. The quantitative Real-Time PCR had higher sensitivity and seropositivity and was considered the best method for the diagnosis of Hepatitis B infection. It was concluded that individuals of these localities are highly burdened by hepatitis B viral infection which should devise proper preventive measures to reduce the risk of infection.