112 aerobic moderate halophilic bacteria were isolated from Daong Ancient Brine Well located in Zigong city, Sichuan Province, China. And they were subjected to the analysis of phenotype, physiology, 16S rRNA sequence. Furthermore, the intra specific phylogeny of closely related strains was also screened by PCR fragment length polymorphism of 16S-23S ribosomal RNA intergenic spacer regions (ISR). The result showed that the halophilic isolates found in current study were closely related to the following genera: Planococcus, Halomonas, Halobacillus, Oceanobacillus and Virgibacillus, a lineage of the domain Bacteria. 16S rRNA analysis revealed that their sequences sharing 100% and 99% similarity were obtained from the GenBank database for P. rifitiensis, H. venusta, H. trueperi, H.blutaparonensis, O. profundus and V. pciturae. However, isolates QW06、QW12、QW15 and QW18 exhibited differences from their corresponding reference strains to some degree, including colony pigmentation, gram staining, acid production and hydrolysis of gelatin, casein and starch. The ISR analysis disclosed variation of banding pattern in these isolates related to Oceanobacillus and Halobacillus which was observed with phenotypic and physiological characterizations as well. It was clear that these halophiles have adapted to the special man-made hypersaline environment by the basic physiological evolution during phylogenesis. So they could be determined according to further polyphasic taxonomy data. The current primary result also indicated the diversity of species of culture-dependent moderately halophilic bacteria colonizing in Dagong Ancient Brine Well as well as phenotypic and genotypic and phylogenetic diversity.