Combined HIV infection can accelerate HBV-induced liver disease. It is known that HBV Pre-S deletion is closely related to HBV-associated terminal liver disease in HBV mono-infection. Currently, data on HBV Pre-S quasispecies feature deletion in HIV/HBV co-infected patients are lacking.The characteristics and blood samples of patients with chronic HBV infection were collected and classified into an HIV/HBV co-infection group and an HBV mono-infection group according to HIV antibody results before treatment. HBV DNA in serum was extracted. The HBV Pre-S region was amplified by nested-PCR and was further T-A cloned. Using the standard sequence of the matched genotype HBV as a reference, BioEdit 7.0 software was employed for sequence alignment.HBV Pre-S regions were successfully amplified from 147 patients, including 71 cases in the HIV/HBV co-infected group and 76 cases in the HBV mono-infected group. The proportion of the HIV/HBV co-infected group with Pre-S quasispecies deletion was lower than that of the HBV mono-infected group. By analyzing the frequency of Pre-S quasispecies in the two groups, the frequency of Pre-S quasispecies in HIV/HBV co-infected patients with Pre-S quasispecies was higher than HBV mono-infected patients. The frequency of Pre-S quasispecies deletion of the S protein promoter region in the HIV/HBV co-infected group was significantly higher than that in the HBV mono-infected group.High-frequency Pre-S quasispecies deletions are predominant in HIV/HBV co-infected patients; however, low-frequency Pre-S deletions are predominant in HBV mono-infected patients, providing a reference for the pathogenesis of the accelerated progression of liver disease in HIV/HBV co-infection.