© 2016 Homs et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Chronic HDV infection can cause a severe form of viral hepatitis for which there is no specific treatment. Characterization of the hepatitis B or C viral quasispecies has provided insight into treatment failure and disease recurrence following liver transplantation, has proven useful to understand hepatitis B e antigen seroconversion, and has helped to predict whether hepatitis C infection will resolve or become chronic. It is likely that characterization of the hepatitis delta virus (HDV) quasispecies will ultimately have similar value for the management of this infection. This study sought to determine the RNA evolution rates in serum of chronic hepatitis delta (CHD) treatment-naïve patients, using next-generation sequencing methods. The region selected for study encompassed nucleotide positions 910 to 1270 of the genome and included the amber/W codon. Amber/W is a substrate of the editing process by the ADAR1 host enzyme and is essential for encoding the 2 delta antigens (HDAg). The amber codon encodes the small (unedited) HDAg form and the W codon the large (edited) HDAg form. The evolution rate was analyzed taking into account the time elapsed between samples, the percentage of unedited and edited genomes, and the complexity of the viral population. The longitudinal studies included 29 sequential samples from CHD patients followed up for a mean of 11.5 years. In total, 121,116 sequences were analyzed. The HDV evolution rate ranged from 9.5x10-3 to 1.2x10-3 substitutions/site/year and showed a negative correlation with the time elapsed between samples (p<0.05). An accumulation of transition-type changes was found to be responsible for higher evolution rates. The percentages of unedited and edited genomes and the quasispecies complexity showed no relationships with the evolution rate, but the fluctuations in the percentages of genomes and in complexity suggest continuous adaptation of HDV to the host conditions.