The Amazon region is considered to be a high en- demic area for Hepatitis B Virus (HBV) infections, Rondônia state having the highest prevalence. The aim of this study was to identify molecular genotypes and mutations in the S gene region of HBV viral ge- nomes from 20 patients using a DNA microarray. Results: Serological tests showed that 88% of patients were HBeAg negative, 82% had anti-HBe antibodies and 33% were co-infected with Hepatitis Delta Virus. Sixteen percent of the patients were considered cir- rhotic, and 11% have been transfused. The microar- ray technique identified the genotypes A (4 patients), D (7 patients) and F (7 patients) in 18 samples. Muta- tions were detected in all 3 genotypes and, overall, A159G, which has been associated to a reduced anti- genicity of the virus, was detected most frequently. In genotype A, G119E was the most frequently detected mutation followed by mutations A159G, F134Y, W172C, Y161F and T143S. A159G was detected in all genotype D and F samples followed by mutations T143S, Y161F, N131T, T114S and G119E in genotype D and mutations T143S, Y161F, N131T, T114S and G119E in genotype F. Conclusion: The analysis of mutations repartition among genotypes suggests that some of them are preferentially or exclusively associa- ted with genotype A, D or F. This type of tool is adapted for clinical and therapy monitoring of pa- tient as well as for molecular epidemiology research on HBV.