The prokaryotic diversity of a non-sulfide, low-salt cold spring sediment was investigated by constructing bacterial and archaeal clone libraries of the 16S rRNA gene. 241 bacterial clones were screened, which could be grouped into 86 ribotypes, based on restriction fragment length polymorphism (RFLP) analysis. These were divided into 11 phyla (Actinobacteria, Acidobacteria, Bacteroidetes, Chlorobi, Cyanobacteria, Firmicutes, Gemmatimonadetes, Nitrospirae, Proteobacteria, Planctomycetes and Verrucomicrobia). Of these, Acidobacteria and Proteobacteria were the most dominant, representing 48% and 25% of the total bacteria clone library, respectively. For the archaeal clone library, 121 positive clones were screened and 22 ribotypes were determined. BLAST analysis indicated that all ribotypes were affiliated with the phylum Crenarchaeota. Phylogenetic analysis classified them into three subgroups (Groups I-III). Groups I and III, belonging to the Soil-Freshwater-subsurface group and Marine group I, respectively, were the dominant groups, representing 50% and 47% of the library, respectively. Of them, 20% of ribotypes were related to the cold-loving Crenarchaeota. These findings show that bacteria in spring sediments are more diverse than are archaea; in addition, the spring harbors a large number of novel bacterial and archaeal species and maybe exist novel lineages.