To understand the impact of land use changes on the composition and structure of fungal communities from Miyaluo county in subalpine forest area of western Sichuan, the molecular diversity of 18S rDNA genes from soil obtained at a 20-year-old spruce plantation (Picea likiandensis var balfourianan) and cropland sites were examined using a PCR-based cloning approach. DNA was directly extracted from the soil microorganisms and amplified the 18S rDNA gene fragment using PCR by the specific primers of EF-4f: 5′-GGAAGGG(A/G)TGTATTTATTAG-3′ and Fung-5r: 5′-GTAAA AGTCCTGGTTCCC-3′. For the gene fragment, diverse PCR products were characterized by cloning, restriction fragment length polymorphism (RFLP) analysis and sequencing. A total of 238 clones and 56 operational taxonomic units (OTUs) which were digested by the restriction enzymes MspI and RsaI were obtained from all samples. The 20-year-old spruce plantation and cropland sites were received 137 and 101 clones and 37 and 19 OTUs, respectively. There were different significant dominant groups of clones occurring in both samples and shared 6 OTUs. 20-year-old spruce plantation and cropland sites had one and two significant dominant groups which account for 20.4%, 25.7% and 21.8% of all clones, respectively. There also were some secondary dominant groups of clones which account for 7.3%-8.9% of all clones. Fourteen 18S rDNA clones were sequenced and their nucleotide identity was from 86% to 99%. Compared the known sequences with the deposited in the data bank (www.ncbi.nlm.nih.gov), their level of nucleotide identity was from 92% to 100%. The phylogenetic tree was constructed by the Clustal W and Mega softwares. 14 sequences could be subdivided into 3 clusters in the phylogenetic tree. The clone sequences of 20-year-old spruce plantation site were completely clustered into the first and the third clusters, and the clone sequences of cropland site only distributed in the second cluster. Therefore, both 20-year-old spruce plantation and cropland sites had the high fungal diversity, and land use changes significant influenced the fungal community and structure.