Brucellosis is an important zoonotic disease caused by different species of genus that are pathogenic for humans and a variety of animals. Accurate detection of spp infection is important for control of disease. The aim of this study was to comparison of molecular genotyping of strains by Pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction -Restriction Fragment Length Polymorphism (PCR-RFLP) techniques. Twenty- seven spp. were isolated from human and animal samples. The isolates identified by conventional microbiological methods and confirmed using PCR for amplification of gene. Molecular typing of strains carried out by PCR-RFLP after and PFGE of chromosomal DNA after enzyme digestion. The gene PCR Products with different patterns of PCR-RFLP were sequenced. The amplification of all human and animal isolates were positive for 1100 bp fragment. By PCR-RFLP analysis two genotypes/patterns for human isolates and four genotypes for animal isolates were obtained. In PFGE analysis totally, 7 common clones/clusters and 3 single clones were obtained. The results of this study showed the PFGE method is the more reliable and useful assay for molecular typing of strains and is more preferred to PCR-RFLP in determination of genetic similarity among human and animal isolates. The presented data showed PCR-RFLP analysis was not able to differentiate between biovars and vaccine strain.