Previous studies have demonstrated limited potential for acclimation of adversely affected olfactory behaviours in reef fishes under elevated CO2, indicating that genetic adaptation will be required to maintain behavioural performance in the future. Adaptation depends on the presence of heritable phenotypic variation in the trait, which may differ between populations and environments. We used parent-offspring regressions to estimate the heritability (h2) of variation in behavioural tolerance to high CO2 (754 μatm) in both field-collected and laboratory-reared families of Acanthochromis polyacanthus. Tolerance to elevated CO2 was measured by determining the behavioural response of individuals to chemical alarm cues. Both populations exhibited high heritability of olfactory behaviour phenotype (father-midoffspring h2 = 0.56 & 0.65, respectively) when offspring were acutely exposed to high CO2 for 4 days. However, there was no heritability in the behavioural phenotype when juveniles were chronically exposed to high CO2 for 6 weeks in the laboratory-reared families. Parental exposure to high CO2 during the breeding season did not alter this relationship between heritability and length of juvenile exposure to high CO2. These results demonstrate that variation in behavioural tolerance to high CO2 is heritable, but adaptive potential may be constrained by a loss of phenotypic variation when juveniles permanently experience a high CO2 environment, as will occur with rising CO2 levels in the ocean.