Aims: To evaluate rpoB gene as a biomarker of microbial biodiversity associated to cold-smoked salmon by a novel nested-polymerase chain reaction/temporal temperature gradient gel electrophoresis (PCR/TTGE) technique applied on pure cultures of reference strains. Methods and Results: DNA obtained from pure cultures of reference strains was used in a succession of a first PCR amplification of rpoB fragment with degenerated nonclamped primers and a nested-PCR with nondegenerated clamped primers. PCR products were then applied on a TTGE gel in order to analyse strains profile. High quantity of nested-PCR products were obtained for each tested strain and TTGE profiles showed a good separation between the different reference bacteria and an easy way to associate one band to one species. Conclusion: The nested-PCR/TTGE technique used in this study is a promising way of investigating bacterial community structure of cold-smoked salmon or other food matrix. Significance and Impact of the Study: Because of its single copy state leading to single band profiles in TTGE, rpoB constitute a good potential molecular marker for further development of cold-smoked salmon biodiversity analysis.