Objective: To determine the frequency of hepatitis B virus (HBV) infection in patients of Non- Hodgkin's Lymphoma (NHL) at tertiary care centers of Larkana. Study Design: This is a Cross sectional study. Study Setting: This study was conducted at Department of Medicine in collaboration with Department of Pathology, Chandka Medical College (C.M.C) Hospital Larkana and Larkana Institute of Nuclear Medicine and Radiotherapy (LINAR), over two years period from March 2008 to February 2010. Material and Method: Total 127 cases of biopsy proven non-Hodgkin's lymphoma, age 5-74 years of either sex visited medical OPD of CMC Teaching Hospital Larkana and LINAR were included, blood samples were taken from participants for detection of hepatitis B surface antigen. Results: Out of 127 cases, male were 78 (61.4%) and female were 49 (38.6%) among them 26 (20.4%) patients were infected by hepatitis B virus. Infected male were 15 (57.7%) and female were 11 (42.3%). The maximum infected patients were in the 41-60 years of age grouping i.e. 11 (42.3%). High grade and stages-1V of NHL patients were infected mostly, 14 (53.8%) and 11(42.3%) respectively. B symptoms were observed in 17 (65.38%) out of 26 patients of infected group in comparison to 60 (59.40%) of non-infected group. Conclusion: This study shows high frequency of hepatitis B virus infection in Non-Hodgkin's lymphoma patients, particularly in higher grade and stage.