In all jawed vertebrates (Gnathostomata) studied to date, one of the hemoglobin gene clusters is linked to the widely expressed Nprl3 gene. Where examined, introns of Nprl3 contain remote erythroid-specific enhancers which activate the linked hemoglobin genes. In sea lamprey (Petromyzon marinus), a jawless vertebrate (Agnatha), one of the hemoglobin clusters is also linked to an nprl3 orthologue suggesting that this regulatory mechanism may have been established in a common ancestor to Agnatha and Gnathostomata. Here, we show that the nprl3-linked hemoglobin locus of the river lamprey Lampetra fluviatilis corresponds to the Gnathostomata Nprl3-linked hemoglobin locus. Functional analysis demonstrates that an erythroid-specific enhancer located in intron 7 of lamprey nprl3 corresponds to the erythroid-specific hemoglobin enhancer (MCS-R1) present in the same intron of mammalian Nprl3. Thus, an nprl3-linked hemoglobin locus, regulated by a remote enhancer driving expression in erythroid cells, was established prior to the divergence of Agnatha and Gnathostomata.