Research on the diversity of microorganism community in natural environment has been concerned hot spot using the newly molecular biotechnology in the world now. This was the first description of the molecular diversity and phylogenetic analysis of nitrogen-fixing (nifH) genes in alp prairie soil of Sanjiangyuan natural reserve. DNA was directly extracted from the soil microorganism and amplified the nifH gene fragment using PCR by the primers of nifH-34F 5'-AAAGG(C/T)GG(A/T) ATCGG(C/T)AA(A/G) TCCACCAC-3' and nifH-491R 5'-TYGTT(G/C)GC(G/C)GC(A/G)TACAT(G/C)G CCATCAT-3'. For the gene fragment, diverse PCR products were characterized by cloning, restriction fragment length polymorphism (RFLP) analysis and sequencing. 143 clones and 35 different RFLP patterns were received in two samples by the restriction enzymes MspI and RsaI digested. ZD sample had 82 clones and 21 different RFLP patterns, and YS sample had 61 clones and 19 different RFLP patterns. There were shared 5 RFLP patterns in two samples. The analysis result found a significant dominant group of clones occurring in both samples which account for 29.3% and 32.8%, respectively, and several minor groups were also detected. 21 clones were sequenced, and their levels of nucleotide identity were from 71% to 98%. None of the sequenced nifH gene was completely identical to any deposited in the data banks, and therefore each of them belong to a noncharacterized bacterium. Finally, the phylogenetic tree was constructed by the Clustal W and Mega software. 21 sequences can be subdivided into 4 clusters in the phylogenetic tree, and most of them had the closely similar toalpha- , beta-, and gamma-Proteobacteria . The significant dominant group in YS sample and ZD sample had the closely related with Rhodobacter sphaeroides and Delftia tsuruhatensis, respectively. The YS-nifH-11 was the only sequence which had highly similar to Cyanobacteria .