FunGAP is a Python-wrapped fungal genome annotation pipeline running under the Linux/Unix operating system. The annotation procedure used in FunGAP requires two inputs, genome assembly and RNA-seq reads. FunGAP aims to predict the most feasible gene from all plausible gene models obtained from various gene prediction programs using multiple strategies such as ab initio, EST-, and/or homology-based methods. This guide covers how to run the FunGAP from the command line and use various options for practical gene prediction. Users can choose options for quality control of the input sequences, selecting model database, filtration of predicted gene models, and post-process such as checking genome completeness and transposable elements. Using FunGAP, the user will acquire a high-quality fungal gene prediction for post-genome sequencing analysis.