Introduction: Hepatitis B virus (HBV) infection and its complications are major public health problems. As it is hard to treat and control the chronic state, control of disease depends on the prevention especially by vaccination. There is an impaired immune response to vaccinations including HBV in patients with some malignancies. The aim of this study is to assess the response rate of patients undergoing chemotherapy to HBV vaccination. Materials and Methods: All patients from two hematology/oncology clinics in Isfahan, Iran with the history of at least 1 month chemotherapy who had the inclusion criteria were enrolled in a case control study. Also a sex- and age-matched control group from healthy population was selected. They were vaccinated in a schedule of 0, 1, and 6 months and were examined for antibody titers 1 month after the last dose. The titers more than 10 mIU/ml were determined as positive response to vaccination. Results: In this study, 50 patients and 50 healthy subjects were enrolled. The two groups were age and sex matched (P > 0.05). Frequency of negative responses to HBV vaccination in case and control groups were 9 (18%) and 1 (2%), respectively (OR = 10.75, CI = 1.30–88.47, P = 0.027). Of 50 patients, 54%, 12%, 22%, and 12% had breast cancer, lymphoma, gastrointestinal, and genitourinary cancers, respectively, and frequency of negative responses were 3 (11%), 1 (16%), 4 (36.4%), and 1 (16%), respectively (P = 0.167). Conclusion: According to our results, malignancy and chemotherapy will have an important effect on the immune system and cause negative response to HBV vaccination. Our results revealed the importance of passive immunity and screening for HBV infection in patients undergoing chemotherapy. Also more studies for better vaccination schedules in this group of patients are recommended.