Bartonella is a significant human pathogen and is the world's most common bacterial zoonosis acquired from companion animals. However, there is no uniform method for Pulse-Field Gel Electrophoresis (PFGE) for Bartonella population genetics studies. Further, some genes of Bartonella can mutate frequently and may affect the use of PFGE for Bartonella. Here we designed methods to solve these problems. We standardized the bacterial concentration, selected the appropriate digestion enzyme, optimized the electrophoretic parameters and characterized reproducibly two Bartonella species strains. Thus we optimized the PFGE procedure and determined how often Bartonella mutated. Our data shows a practical protocol for inter- and intra-species identification of Bartonella and was reproducible using two species strains that showed no mutation occurred after two passages for B. elizabethae; but mutation did occur in B. henselae.