Hepatitis B virus (HBV) genetic variability, shaped by high mutation rates and selective pressures, complicates its management and increases the emergence of drug-resistant and immune-escape variants. This study aims to analyze immune escape mutations (IEMs) and drug resistance mutations (DRMs) in patients with HBV infection exposed to antiviral therapies and exhibiting detectable plasma HBV viremia. This monocentric retrospective real-life study was carried out at the ASST Spedali Civili di Brescia, Italy, from 2015 to 2023. A total of 102 consecutive patients with detectable serum HBV-DNA exposed to at least one NA and for whom a drug resistance assay was available were included in our study. HBV sequences were amplified, sequenced, and analyzed for mutations using Geno2pheno and Stanford University tools. Phylogenetic analysis and statistical regression were performed to confirm genotypes and identify mutation patterns and associated risk factors. Our study shows a 38.2% prevalence of DRMs, with M204I/V (95%) and L180M (64%) being the most common, and a 43% prevalence of IEMs, primarily in the major hydrophilic region. Genotype D3 exhibited a higher mutation burden than other genotypes. Significant associations were found between HBsAb presence and increased IEM burden, while HBeAg was protective against DRMs. Atypical serological profiles were observed in 18.6% of patients, including cases of HBV reactivation under immunosuppressive therapy. This study highlights the high prevalence of IEMs and DRMs in a real-world setting, particularly among HBV genotype D3 carriers. These findings underscore the importance of mutation surveillance to guide therapeutic strategies, vaccine design, and public health policies to address the challenges posed by HBV genetic variability.