We describe a new technique for transgenerational marking of embryonic otoliths that promises significant advancements in the study of larval dispersal and population connectivity in marine fishes. The approach is based on maternal transmission of 137Ba from spawning females to egg material that is ultimately incorporated into the otoliths of embryos produced by an individual after exposure to the isotope. We injected females of a benthic-spawning clownfish (Amphiprion melanopus) and a pelagic-spawning serranid (Centropristis striata) with enriched 137BaCl2 and then reared the resulting progeny through to settlement. Barium isotope ratios in the cores of larval otoliths were quantified using laser ablation inductively coupled plasma mass spectrometry. Larval otoliths from both species contained unequivocal Ba isotope signatures over a wide range of doses (0.8?23 µg 137Ba·g female?1). Female A. melanopus continued to produce marked larvae over multiple clutches and for at least 90 days after a single injection. The ability to administer different combinations of stable Ba isotopes provides a new means of mass-marking larvae of benthic- and pelagic-spawning fishes from multiple populations over extended spawning periods.