Fungi have critical roles in many ecosystems. They can be a threat to the health of humans and cause structural integrity issues in environments. Since most fungal diversity is unknown, there have been new methods to improve phylogenetic and non-phylogenetic analysis. The development of PCR primers for the Internal Transcribed Spacer (ITS), combined with next generation sequencing, has significantly improved the ability to profile fungal diversity of microbes. The high sequence variability within the ITS region allows for more accurate species identification. However, high variability makes multiple sequence alignment and phylogenetic analysis significantly less accurate between distant fungi. To solve this issue, a bioinformatics tool (ghost-tree) was applied. Ghost-tree integrates sequence data from two genetic markers into one phylogenetic tree, which can be used for reliable phylogenetic analysis. The main theme of the project is to determine if there are differences between these four fungal communities. In addition, it is a goal to determine which environments differ most significantly and the species driving these differences among communities. The alpha-diversity test concluded that the soil community had the greatest amount of different fungal species in one sample. Then, a beta-diversity test (UniFrac method) was applied and resulted in a 3D PCoA plot for visualization. A Kruskal-Wallis test was used to see if there were differences of means among the four environments. Then, a PERMANOVA test was conducted to determine environments that differ the most and which species made these environments different. By using pair-wise tests, it was determined that 4 groupings are significantly different from the environment being compared against: Leaf/Rhizosphere (p-value = .003), Leaf/Soil (p-value = .007), Insect/Rhizosphere (p-value = .009), and Insect/Leaf (p-value = .013). Focusing on Leaf/Rhizosphere, few species contributed a significant percent to the differences among the Leaf and Rhizosphere environments. It can be concluded that there are significant differences among the four communities, unknown species contribute significantly to these differences, and soil has the greatest fungal diversity within a sample. By increasing taxonomic resolution, further studies can focus on microbial shifts among fungal communities.