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Oocyte hydration is a unique event in oviparous marine teleosts that provides the single-celled egg with an essential pool of water for survival during early development in the saline oceanic environment. A conserved mechanism of maturational yolk proteolysis of a neofunctionalized vitellogenin (VtgAa) has been shown to underlie the hydration event in all teleosts that spawn pelagic eggs (pelagophils), and is argued to be a key adaptation for teleost radiation in the oceanic environment 55 Ma. We have recently shown that a small pool of free amino acids (FAAs) significantly contributes to the osmolarity of the ovulated egg in an ancestral marine teleost, the Atlantic herring that spawns benthic eggs (benthophil). To determine whether multiple forms of vtg exist and whether neofunctionalization of the gene products are related to the egg FAA pool in this species, genomic sequences conserved between the exons of Atlantic herring and zebrafish were amplified. This approach identified a small polymorphic intron between exons 9 and 10 in Atlantic herring and demonstrated that two closely related major vtg transcripts (chvtgAc1 and chvtgAc2) are expressed during oogenesis. A separate polymerase chain reaction-based approach identified a more ancestral phosvitinless transcript (chvtgC). Proteomic analyses of the translated products of the major vtg forms demonstrated that the yolk proteins are similarly processed during deposition, and oocyte maturation and reveal that vtgs have duplicated but not neofunctionalized in this species. Phylogenetic analyses consistently clustered the transcripts and proteins as the basal sister group to the Ostariophysi in full congruence with the Clupeocephalan rank, and suggest that expansion of ostariophysan vtgAo1 and vtgAo2 genes occurred in a lineage-specific manner after separation from the Clupeiformes. Three-dimensional modeling of the ChvtgAc1 sequence against the resolved lamprey lipovitellin module revealed that the tertiary structure is highly conserved, with most substitutions occurring on the outside of the molecule. The data indicate that the phosvitin domain, the smallest yet reported for teleosts, and an N-terminal fragment of the lipovitellin light chain contribute to the FAA pool. The present findings thus show that yolk proteolysis and the generation of an organic osmolyte pool of FAAs was an adaptive response to spawning in seawater also for the Clupeiformes, but that this process was not evolutionarily successful in terms of biodiversity until vtg gene neofunctionalization occurred in the Acanthomorpha.