This chapter aims to introduce the workflow for fungal biodiversity investigation, especially focusing on DNA metabarcoding, which is the predominant method used for studies. It explains what the molecular method and DNA metabarcoding are, and briefly review the studies investigating fungal diversity in polar regions using DNA metabarcoding. The chapter analyses the workflow of experiments and analyses of fungal diversity using DNA metabarcoding. It discusses the prospects for diversity studies using DNA metabarcoding. The chapter presents an example in which MiSeq is used. It introduces only studies which focused on the taxonomic diversity. The sampling design and strategy are important points in testing one’s hypotheses about ecological systems. For example, sampling methods, sampling effort, and sequencing depth have been reported to affect the results of diversity investigations and community analyses. After sampling, the collected samples must be preserved with caution to prevent DNA degradation and changes in the fungal community composition.