The bacterial composition and diversity in Xinjiang Shawan cold spring sediment were investigated by direct extracting environmental total DNA and constructing clone libraries of the 16S rRNA gene amplified with bacteria-specific primers. 241 bacterial clones were screened, which could be grouped into 86 Operational Taxonomic Units (OTUs), 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) by phylogenetic analysis. Of these, Acidobacteria and Proteobacteria were the most dominant, representing 48% and 25% of the total bacteria clone library, respectively. More than 1/3 of the total OTUs showed less than 95% of sequence identity with sequences deposited in GenBank database. In addition, nearly 20% of clones were related to nitrogen-fixing and nitrite-oxidizing bacteria. These results suggested that bacteria in the spring sediment were highly diverse in species richness and maybe existence of large number of unknown groups.