Mushrooms and insects are both integral components of ecosystems worldwide. Interactions between these two groups of organisms are particularly interesting to study due to the transient nature of mushroom fruiting bodies, which form for a short period of time when environmental conditions are optimal to allow the fungus to distribute its spores. Despite this unpredictability in where and when mushrooms will grow, a variety of insect species use mushrooms as a food source and a substrate on which to lay their eggs. Interactions between these two groups of organisms have been documented extensively in Europe. However, little is known about the diversity of insects that associate with mushrooms in Canada. In this study, 1,017 mushrooms were collected from forests in Hamilton and the Tillsonburg, Ontario regions between fall 2018 and fall 2019. Mushrooms and their associated insects were identified through DNA barcoding using sequences of the nuclear ITS region and the mitochondrial CO1 gene for the mushrooms and insects, respectively. In total, more than 100 insect species from at least 35 families and five orders were identified from the approximately 200 mushroom species collected. While some insect species displayed evidence of specificity in their choice of mushroom host, the larger network of associations was moderately generalized and many insect species inhabited mushrooms from multiple families and orders. This study highlights the incredible diversity of organisms that rely on mushrooms for survival and contributes to our overall understanding of mushroom-insect associations in this region of Southern Ontario. / Thesis / Master of Science (MSc) / Mushrooms and insects are both highly important organisms within ecosystems around the world. Mushrooms play key roles in breaking down organic matter within forests and contributing to plant health, while insects are important decomposers and pollinators. This project involved collecting over 1,000 mushrooms from two regions in Southern Ontario and identifying the insect species found living within these mushrooms in order to examine patterns in the interactions. To our knowledge, this is the first time a survey of mushroom-insect associations has been conducted in Canada. The collected mushrooms were inhabited by a diverse range of insect species, which included mostly flies and beetles, and each of the main sampling locations had their own unique mushroom and insect communities. Some insect species displayed preference in their choice of mushroom host. Overall, this study contributes greatly to our current understanding of mushroom and insect diversity in this part of Canada.