The mutation rate of the hepatitis B virus (HBV) is often obtained by statistical software. This is restricted to the sampled data and cannot be applied to other cases. This limitation makes an equation which can accurately predict the mutation rate both necessary and important. The goal of this paper is to derive an equation for estimating the mutation rate of the hepatitis B virus. The derived equation is based on the JC (Jukes and Cantor) method and further takes polymorphism mutation into consideration to estimate the evolutionary rate after one year and 40 years from the sampled data over 4 months and 12 months, respectively. The paired t-test is used to evaluate the degree of similarity between the results of the proposed equation and the real evolutionary rate (P-value is 0.32). The 40 year results are also comparable with related research. These results demonstrate that the proposed equation would be a useful technology to the HBV sequence phylogenetic analysis of evolutionary...