Identification and classification of a wide variety of organisms using DNA sequences has helped overcome many limitations of traditional morphological approaches. However, the utility of DNA as a tool to catalogue biodiversity, resolve phylogenies, or explore patterns in ecological communities depends strongly on choice or design of primers for selecting the appropriate genetic markers. Unfortunately, no primers have been developed to target the D1 region across a wide diversity of fungi, with a short amplicon suitable for the Illumina MiSeq platform. Here we introduce and evaluate two primer sets targeting the D1 region of the large subunit of ribosomal DNA to evaluate fungal diversity in 24 environmental subarctic soil and peat samples.