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Background Current knowledge about genetic diversification within the HDV ribozyme (critical for viral replication) and HDAg domains, which drive replication and contain cytotoxic T lymphocyte epitopes (CTLEs), remains limited. We examined the degree of genetic conservation in HDV ribozyme and HDAg regions and evaluated their relationship with virological and biochemical markers.Material and Methods 178 individuals with chronic HDV infection were enrolled. Full-length HDV genomes were sequenced by Illumina (median [IQR] reads/patient: 69,282 [38,127–181,165]). Genotypes (Gt) were assigned by phylogeny. Nucleotides and amino acids were considered conserved if not mutated in ≥99% of sequences. HDAg domains and CTLEs (N=18) were defined according to Pascarella 2010 and Kohsar 2021.Results Individuals were 58% males, median age 51 (37-59) years, mainly Italians (ITs, 43.7%), followed by Eastern Europeans (EEs, 34.8%) and Africans (AFs, 21.5%). Median HDV-RNA and ALT levels were 5.4 (4.5-6.1) logIU/ml and 94 (62-151) U/l. The most prevalent Gts were 1e (57.8%) and 1c (20.8%), followed by 1b (9.5%), 1a (7.8%), 5 (2.2%), 7 (1.1%) and 6 (0.5%). Gt 1e was predominant in ITs (64%, P<0.001), 1c in EEs (81%, P<0.001), while 1a, 1b, 5, 6, and 7 were found only in AFs.HDV ribozyme showed 100% nucleotide conservation whereas HDAg exhibited marked variability:conservation was lower in N-terminal (aa1-107) than in C-terminal domains (aa108-214, known to be crucial for HDAg activity) (14.7% vs 32.2%, P<0.005). CTLE conservation ranged from 0% in CTLEaa26-34 and CTLEaa43-51 to 50% in CTLEaa140-149.In Gt 1e, the aa conservation was significantly reduced in individuals >40 years (68.2% vs 30.8%, P<0.0001), particularly within 6 CTLEs. The N12G mutation (median intra-patient prevalence 99%) correlated with higher ALT (101[63-152] vs 86 [69-174] U/l, P=0.01) and was enriched in individuals with 2xULN ALT (77% vs 50%, P=0.005), suggesting enhanced cytolytic activity.Conversely, T180A (median intra-patient prevalence 63%) correlated with lower HDV-RNA and HBsAg levels (4.9 vs 5.6 logIU/ml, P=0.04 and 3.7 vs 3.9 logIU/ml, P=0.03), indicating impaired viral fitness.Conclusions HDAg exhibits conspicuous genetic diversification, particularly in N-terminal domains and CTLEs, with progressive enrichment in older individuals, suggesting ongoing antigenic drift. Specific mutations may modulate viral fitness and HDV-driven cytolytic activity, warranting further in vitro studies. The highly conserved ribozyme represents a promising target for RNA interference-based therapeutic strategies.