According to the extensive distribution of GYF domain proteins and the diversity of its functions, it is necessary tostudy GYF domain proteins further and conduct evolutionary analysis. Since AtEXA1 is the most well-studied GYF domain proteinin plants, this study mainly focuses on the proteins similar to AtEXA1. Analysis of 8 053 GYF domain proteins in eukaryotes ranging from unicellular lower fungi to higher organisms showed that AtEXA1-like proteins are mainly found in plant species, whileGYF proteins from animals have low similarity to AtEXA1. It was also found that AtEXA1-like proteins are present in almost allagricultural crops. This study demonstrates the conservation of all GYF domain proteins, initially deconstructs the evolution of GYFdomain proteins, and provides implications for subsequent studies on the functional characteristics of GYF domain proteins.