Fusarium is a large and diverse genus of fungi with various ecological characteristics, from saprophytes to mycotoxin producers, phytopathogens but also biocontrol agents and opportunistic human pathogens. It is often important to identify the Fusarium present in an environment to species-level, since they have different life cycles, host range, mycotoxin profiles and climatic preferences. Widely used barcodes for the study of fungal communities such as the ITS do not provide species level resolution for Fusarium. In this project we evaluated Fusarium-specific primers targeting the transcription elongation factor (EF-1a) using mock Fusarium communities and field samples. The aims of the study were 1) to evaluate the accuracy of the primers in reflecting the quantitative relationships among species 2) apply high-throughput sequencing of EF-1a amplicons to study Fusarium diversity 3) to evaluate the method’s performance on more complex environmental material.