Abstract: Pisolithus tinctorius is an important forest ectomycorrhizal fungus with wide application in root stimulations. Nitrate reductase gene, named as ptnr (GenBank accession number KC698974), was cloned from P. tinctorius, using reverse transcript-PCR(RT-PCR)and rapid amplification of cDNA ends(RACE). The annotated ptnr cDNA had a total length of 2 951 bp, with a 2 754 bp ORF, coding 917 amino acids. The encoded protein was estimated at the length of 102.06 ku and with an isoelectric point of 6.34. Both sequence similarity retrieval and phylogenetic tree analyses showed the amino acid sequence homology between P. tinctorius encoded nitrate reductase and Auricularia delicate encoded reductase was the highest, which could reach 60%, while the nitrate reductase sequence homology between P. tinctorius and Coprinopsis cinerea, or ectomycorrhizal fungi Hebeloma cylindrosporum, or Laccaria bicolor is 57%, 53% and 56%, respectively. Our study provides a basis for further research of genes involved in the symbiotic association between ectomycorrhizal fungi and forest.