Fungal glucuronoyl esterases (FGEs) catalyze cleavage of the ester bond connecting a lignin alcohol to the xylan-bound 4- O -methyl- d -glucuronic acid of glucuronoxylans. Thus, FGEs are capable of degrading lignin-carbohydrate complexes and have potential for biotechnological applications toward woody biomass utilization. Therefore, identification and characterization of new FGEs are of critical importance. Firstly, in this study, we built a phylogenetic tree from almost 400 putative FGEs obtained on BLAST analysis and defined six main clades. In the phylogenetic tree, all the putative FGEs of ascomycetes cluster in clades I to IV, and most of the putative FGEs of basidiomycetes (B-FGEs) cluster in clades V to VI. Interestingly, several B-FGEs were found to cluster in clade II; most FGEs of clade II were found to have higher theoretical isoelectric points than those in the other five clades. To gain an insight into the putative FGEs in the clades that have not been characterized yet, we chose the FGEs of Ceriporiopsis subvermispora ( Cs GE) and Pleurotus eryngii ( Pe GE), which belong to clades V and II, respectively. The catalytic domains of both Cs GE and Pe GE were successfully expressed using Pichia pastoris , and then purified. Benzyl glucuronic acid was used as a substrate to confirm the activities of the Cs GE and Pe GE, and the hydrolyzed product, glucuronic acid, was quantified spectrophotometrically. Both Cs GE and Pe GE clearly exhibited the esterase activity. Additionally, we demonstrated that Pe GE exhibits high tolerance toward several denaturing agents, which may make it a potentially more applicable enzyme.