101 isolates of Brucella spp. including the reference (n=7) and the Iranian field strains belonging to last years 1961-2003 (n=94) were biotyped and classified into 9 biotypes. Random amplified polymorphic DNA (RAPD) analysis using a ten mer-AP4 primer was generated 72 reproduciable DNA band. The typeability was 100%. The thirteen isolates were classified into five groups, each containing 2-4 biotypes. The high DNA polymorphisms among the strains suggested that animals in Iran have been infected with genetically diverse isolates however, in the last forty years B.abortus biotype A3 and B.melitensis biotype M1 have been predominantly isolated from brucellosis cases. The results indicated that the RAPD analysis using AP4 primer can be used to attain reproducibility amplify random fragments of DNA from Brucella genomes in order to differentiate at species and sub-species for epidemiological purpose.