Abstract Oct4 is one of the key pluripotent factors essential for embryonic stem cells and induced pluripotent stem (iPS) cells. Oct4 belongs to the POU domain family, which contains multiples genes with various important functions. Although the function of Oct4 has been extensively studied, detailed comparison of Oct4 with other POU family genes and their evolutionary analysis are still lacking. Here, we systematically identified POU family genes from lower to higher animal species. We observed an expansion of POU family genes in vertebrates, with an additional increment in mammalian genomes. We analyzed the phylogenetic relationship, tissue specific expression profiles and regulatory networks of POU family genes of the human genome, and predicted the putative binding microRNAs of human POU family genes. These results provide the first comprehensive evolutionary and comparative analysis of POU family genes, which will help to better understand the relationships among POU family genes and shed light on their future functional studies.