In this study, we systematically identify Old Yellow Enzymes (OYEs) from a diverse range of economically important fungi representing different ecology and lifestyle. Using active site residues and sequence alignments, we present a classification for these proteins into three distinct classes including a novel class (Class III) and assign names to sequences. Our in-depth phylogenetic analysis suggests a complex history of lineage-specific expansion and contraction for the OYE gene family in fungi. Comparative analyses reveal remarkable diversity in the number and classes of OYE among fungi. Quantitative real-time PCR (qRT-PCR) of Ascochyta rabiei OYEs indicates differential expression of OYE genes during oxidative stress and plant infection. This study shows relationship of OYE with fungal ecology and lifestyle, and provides a foundation for future functional analysis and characterization of OYE gene family.