To determine HBV QA complexity, three parameters were used: Shannon entropy, mutation frequency, and nucleotide diversity. The high correlation between the results obtained with these parameters indicates that Sn, Mf and Pi equally represented HBV QA complexity. In addition, MfAA was used to independently explore variability at the amino acid level in both the preCore and Core regions. We found significant differences in the QA complexity parameters according to HBeAg status, with greater complexity in HBeAg- than HBeAg+ samples, in agreement with previous studies. However, the higher complexity seen in HBeAg- cases lacked significance when analyzing QA complexity attending to HBeAg evolution, likely because of the small number of HBeAg-negative patients. Interestingly, both the HBeAg- and HBeAg+/2 groups showed significantly higher QA complexity than HBeAg+ patients when complexity was analyzed at the amino acid level in the preCore and Core regions separately. Therefore, our data provide evidence that increased viral diversity is associated with HBeAg seroconversion and strongly suggest significant evolutionary enhancement that was even more evident in fluctuating HBeAg status. These findings may indicate an increase in evolutionary pressure due to a more intense immune response in HBeAg-negative status, likely associated with the lack of HBeAg and its immunomodulatory Silmitasertib effect. Overall, there were no significant differences in QA complexity between A and D genotypes in the 30 samples analyzed, despite the constraints on main preCore mutation selection and HBV genotype. Moreover, no correlation was observed between ALT levels and QA variability. Although it is assumed that ALT status provides an estimate of the strength of the immunological response against viral infection, ALT can be influenced by many factors and a single point measurement may not be indicative of the long-term immune status of a host. Furthermore, the aim of this study was to sequentially analyze HBV QA complexity with deep clonal sequence coverage to guarantee the complexity calculations, and because of the huge amount of data involved, only ten patients were included. The negative correlation between HBV DNA levels and QA complexity in the present study agrees with recent findings. Although the correlations did not achieve statistical significance, the three main QA complexity parameters showed that the higher the HBV DNA level, the lower was QA complexity in preCore/Core. However, it should be remembered that all samples included had a high viral load. This potentially confounding factor may have contributed to the absence of significance in the correlation studies. Nonetheless, in the separate analysis of the preCore and Core regions at the amino acid level, the significant negative correlation with HBV DNA observed in preCore MfAA suggests that the preCore mutated variants could confer a decrease in preCore fitness to regulate HBV replication.