Several biochemical and virological parameters should be monitored during natural disease evolution and particularly, during antiviral treatment. One of these, HBeAg expression, is associated with a differing course of infection and with the probability of response to antiviral therapy. The presence of HBeAg in serum depends on variants located in the preCore region or in the basic core promoter. In addition, the Core gene BU 4061T contains epitopic domains that play a central role in the immune response against the virus. Therefore, the preCore/Core is an optimal region to investigate QA evolution in relation to host immune system stimulation. In addition, the preCore/Core regulates HBV replication and includes the only non-overlapping sequence in the HBV genome. The evolution of the preCore/Core regions under NUC treatment has been little investigated. Studies involving molecular cloning of this region have reported that HBeAg seroconversion is associated with increased viral diversity. There is also a recent study in which the preCore/Core was analyzed by UDPS using new bioinformatic tools. The aim of this study was to evaluate associations between HBeAg status and HBV QA complexity in the preCore/Core region, and to explore QA complexity under natural evolution and under NUC antiviral treatment. To this end, we UDPS-analyzed HBV variability and QA complexity in the preCore/Core region at baseline, during a period before starting NUC treatment, and during a period of treatment nonresponse. The HBV QA composition and the changes that occur over QA evolution are important factors related to controlling and treating chronic HBV infection. Hence, acquiring accurate knowledge of HBV QA complexity is currently a major challenge for managing chronic hepatitis B patients. Recent reports support the concept that QA complexity is a clinically relevant factor in the course and prognosis of this disease and in the response to treatment. In this line, HBV QA complexity has been associated with the antiviral response in ETV-treated patients, in whom lower complexity was seen in responders than in partial responders. Cheng et al. reported higher viral diversity in HBeAg natural or treatment-induced seroconverters than in non-seroconverters, thereby providing evidence that increased viral diversity is associated with HBeAg seroconversion, in agreement with our observations in the present study. Although these studies have provided valuable findings, the techniques used analyzed only small numbers of clones, and the results may not be representative of the overall viral population, which contains billions of particles. In contrast, next-generation sequencing methods, particularly UDPS used in the present study, enable clonal analysis of thousands of sequences in a single sample, provide a number of clonal sequences to lend reliability to calculation of QA complexity parameters. In this study, we applied UDPS to determine QA complexity in the HBV preCore and Core regions.