Abstract: Heat shock protein (hsp) is highly conserved, that serves a wide range of function in protein folding and transport. It protect from various type of stress including heat shocks. However, it is well known that the virulence of B. melitensis is more than B. abortus, but there is not any strong evidence to verify it. For this purpose, in refer to potent antigenicity of hsps in various infectious as well as some hsp molecules act as potent activator of macrophage (danger signal), we hypothesized that difference in virulence between B. abortus and B. melitensis may be originated from difference in pattern of response to heat shock induced by high degree of fever that usually present in brucellosis. To this end, five B. abortus and five B. melitensis strains isolated from cows and human, were subjected to 39, 40 and 42 ° C heat shocks. The bacterial whole cell proteins were extracted and resolved by SDS-PAGE. Western blotting was used to detect antibody production against the extracted bacterial proteins especially hsp60 in both control and patient sera. SDS-PAGE gels revealed protein bands mainly in the range of 10-100 kDa. The amounts of a 60 kDa protein band (hsp60) was significantly enhanced following heat shock at 42 ° C in relation to the unheated cells in both bacterial species. The heat shock responses in B. abortus and B. melitensis point to the higher production of a 60 kDa protein (hsp60) in both bacterial species, especially in B. abortus. It seems that, lower hsp60 production by B. melitensis would induce a relatively much lower immune response against the bacterium leading to its greater virulence potentials; the sera from Brucellosis patients reacted with several of these cell derived protein bands in western blots, none of which were reactive with sera from healthy individuals. The western blot protein bands showed striking differences. This observation points to the immunogenic properties of hsps, specially the overwhelming response to hsp-60. Therefore, hsp-60 can be a good antigenic candidate for engineering subunit vaccine against Brucella, as well as for ELISA test development.