A promising new method of marking larval freshwater fishes with enriched stable isotopes by means of injecting the maternal parent with the marking agent was investigated. The 138Ba:137Ba ratios in the otoliths of larval golden perch Macquaria ambigua were compared to determine the effect of injecting female broodstock with different dosages of enriched 137Ba at various times before spawning. There was 100% mark success in the progeny of fish injected with 20 μg g−1 of enriched 137Ba 24 h before inducing spawning with hormones and 40 μg g−1 administered at the same time as inducement of spawning. Injection of 40 μg g−1 enriched 137Ba 21 days before spawning resulted in only 81% mark success and suggests rapid elimination of barium in M. ambigua. Injection with enriched 137Ba did not significantly affect the fertilization rate, number of fertilized eggs or hatching rate compared with long-term hatchery records. These results suggest that transgenerational marking is an effective and affordable means of mass-marking larval fishes. Thousands of larval fishes can be permanently marked with a unique artificial isotopic mark via a single injection into the maternal parent, thus avoiding the handling of individual fishes or having to deal with chemical baths. Because no single mark or tagging method is suitable for all situations, transgenerational marking with enriched stable isotopes provides another method for researchers and managers to discriminate both hatchery-reared and wild fishes.