The organophosphate chlorpyrifos, and its active metabolite chlorpyrifos-oxon (CPO), have been attributed to a number of neurodevelopmental disorders. It is unclear if the adverse effects associated with developmental exposure to the active CPO persist into adulthood and future generations. The goal of this study was to investigate whether CPO-associated changes in embryo-larval zebrafish (ZF) behavior at the F0 5 dpf were manifest throughout the life of the exposed F0, and are inherited by subsequent generations. For this study, embryos were exposed to chlorpyrifos-oxon at the environmentally relevant concentration of 0.01 μg/L and a high concentration of 50 μg/L starting at 4 hpf to 5 dpf, and then raised to F2. There was a significant decrease in distance traveled with 5 dpf F0 ZF exposed to the 50 μg/L CPO, with alterations in noncholinergic genes CFOS and LINGO, and alterations in global DNA methylation. CPO-related behavioral effects were ameliorated by day 21 through the F1 generation. This trend changed with hyperactive behavior, increase acetylcholine concentration in F2 zebrafish that were exposed to 50 μg/L CPO during the F0 development. There was also an increase in AChE activity and hypermethylation in F2 0.01 μg/L exposure larvae, indicating that even low dose exposures can have transgenerational effects. Results from this study demonstrate that early life stage exposures to CPO can lead to epigenetic changes in neurological activity, which may lead to alterations in response to CPO in future generations. Abstract summary: This study identified a correlation between CPO exposure during F0 development and significant differences in F2 behavioral, AChE activity and neurotransmitter concentration.