Introduction. Ten viral genotypes (A-J) have been described for hepatitis B virus (HBV) distributed in all the continents. One of the methodologies for determining the viral genotype is restriction fragment length polymorphism (RFLP), a simple and relative not expensive method, but with some limitations. Objective: The initial objective of the project was to identify the HBV genotypes by RFLP in serum samples obtained from patients and blood donors. Although, considering the discrepancies of RFLP patterns it was necessary to perform phylogenetic genotyping and in silico analysis of HBV sequences. Materials and methods: Fifty-six serum samples were obtained. DNA extraction was followed by PCR amplification of a fragment of HBV ORF S. PCR products were then analyzed by RFLP with AlwI, BsrI, CfrI, HpaII and StyI , and some were also sequenced. Patterns obtained were compared with previous reports. I n silico RFLP analysis was performed since differences between the expected and obtained patterns were found. Results: Genotypes A and F, subgenotype F3, were identified in samples. This result is in according to the previous studies carried out in Colombia; indeed, subgenotype F3 is the most frequent in Andean region of the country, while genotype A is the most frequent HBV genotype in the Western region (Choco State) of Colombia. Based on in silico analysis of 229 HBV sequences from GenBank and 11 sequences of this study, we identified the RLFP pattern for genotype F, subgenotype F3, and we described some modifications of genotype A RFLP patterns. Conclusions: The pattern by single nucleotide polymorphism was identified for genotype F, subgenotype F3, by in silico analysis and sequencing. Further robust in silico analysis are necessary for validate the RFLP patterns of HBV genotype and subgenotypes.