Abstract: The AT-hook is a small motif in DNA-binding protein that was first described in the high-mobility-group non-histone chromosomal protein HMG-I/Y. AT-hook family proteins play an important role in chromatin structure assembly, specific binding to target cells, transcription regulation and development regulation in other organisms. We identified 45 AT-hook genes from the rice genome by bioinformatics, analyzed the gene structure and chromosomal distribution, and examined the phylogenetic relationships among rice AT-hook proteins, finally constructing an unrooted tree from alignment of their full-length protein sequences in rice and Arabidopsis. AT-hook proteins grouped into 5 distinct clades (A–E), with no differences in structure or characteristics. The family members were recruited by chromosome replication. Digital gene expression analysis revealed that AT-hook genes mainly express in young panicles. We verified some of the gene expression by qRT-PCR.