Abstract Precise spatio-temporal expression of genes in organogenesis is regulated by the coordinated interplay of DNA elements such as promoter and enhancers present in the regulatory region of a given locus. POU1F1 transcription factor plays a crucial role in the development of somatotrophs, lactotrophs and thyrotrophs in the anterior pituitary gland, and in maintaining high expression of growth hormone, prolactin and TSH. In mouse, expression of POU1F1 is controlled by a region fenced by two CTCF sites, containing 5 upstream enhancer elements, designated E-A (5’ to 3’). We performed comparative sequence analysis of this regulatory region and discovered that three elements, B, C and E, are present in all vertebrate groups except Agnatha. One very long (>2kb) element (A) is unique to mammals suggesting a specific change in regulation of the gene in this group. Using DNA accessibility assay (ATAC-seq) we showed that conserved elements in anterior pituitary of four non-mammals are open, suggesting functionality as regulatory elements. We showed that, in many non-mammalian vertebrates, an additional upstream exon closely follows element E, leading to alternatively spliced transcripts. Here, element E functions as an alternative promoter, but in mammals this feature is lost, suggesting that conversion of alternative promoter to enhancer could be one evolutionary mechanism for enhancer birth. Our work shows that regulation of POU1F1 changed markedly during the course of vertebrate evolution, use of a small number of enhancer elements combined with alternative promoters in non-mammalian vertebrates being replaced by use of a unique combination of enhancers in mammals.