The prokaryotic microbial diversity of the ancient salt deposits in the Kunming Salt Mine, P. R. China was investigated using PCR-DGGE and rRNA approaches. Total community DNA was extracted and purified by a direct method, which yielded amplified DNA of high molecular weight for samples. A variable region of 16S rRNA gene was then amplified by PCR with bacterial and archaeal primers and analyzed by denaturing gradient gel electrophoresis (DGGE). Twenty-seven major bands were detected in the bacterial DGGE profile of the sample, but only one band of pure culture strains of bacteria isolated from the Kunming Salt Mine matched with one band of sample. No band of pure culture strains of archaea isolated from the Kunming Salt Mine matched with 18 major bands of sample. The results indicated that most of microbes in this environment are likely uncultivable. Clones on the plate were not the predominant species in the community. Two 16S rRNA gene clone libraries (bacteria and archaea) were also constructed, and 36 and 20 clones were selected for amplified ribosomal DNA restriction analysis (ARDRA). ARDRA with enzymes AfaⅠ、HhaⅠ、HaeⅢ revealed 10 bacterial operational taxonomic units (OTUs), with three most abundant OTUs accounting for 38.9%, 25.0%, 16.7% of all the bacterial 16S rDNA clones, respectively. The remaining 7 OTUs presented at low levels, were represented by a single clone. Eight archaeal OTUs were obtained but no predominant OTUs. Some clones were sequenced and each sequence was compared with all nucleotide sequences in GenBank database. Examination of 16S rDNA clones showed that the ancient salt deposits in the Kunming Salt Mine contained a phylogenetically diverse population of organisms from the Bacteria domain with members of three major lineages represented: α-proteobacteria, γ-Proteobacteria and Actinobacteria, especially Pseudomonas. Surprisingly, we recovered a variety of sequence closely related to Actinobacteria which was not found in other salt deposits. All of archaeal clones are from Halorubrum, Haloterrigena and uncultured archaea. The results of DGGE and clone library profiling analysis both indicated that microbial community of the Kunming Salt Mine had higher diversity. In this initial survey, our polyphasic approaches demonstrated that novel and uncultured microbes thrive in the ancient salt deposits of the Kunming Salt Mine. Molecular analysis of the microbial diversity in salt deposits provides foundation for better application of microbial resources.