Chlorpyrifos, a broad‐spectrum neurotoxic organophosphate insecticide, is subject to atmospheric and hydrolytic transport from application sites to aquatic ecosystems. Across the landscape, concentrations in surface water can vary spatially and temporally according to seasonal use practices. Standardized bioassays can provide a screening‐level understanding of aquatic receptor acute and chronic toxicity. However, these bioassays do not address ecologically relevant exposure patterns that may impact fitness and survival within and across generations. The aim of the present study was to evaluate the utility of a second‐tier, screening‐level methodology employing Daphnia magna multi‐ and transgenerational bioassays spanning four generations to investigate the effect of variable chronic chlorpyrifos exposure. The multigenerational assay consisted of continuous chlorpyrifos exposure across four consecutive 21‐day bioassays using progeny from the previous assay for each successive generation. In the transgenerational assay, only the parent (F0) generation was exposed. For both assays, survival and reproduction were assessed across treatments and generations. Results indicated that (1) following continuous chlorpyrifos exposure at ecologically relevant concentrations to four generations of D. magna, the highest treatment showed an apparent tolerance response for both survival and reproductive success in the F3 generation, and (2) chlorpyrifos exposure to the F0 generation did not result in treatment effects in the unexposed F1, F2, and F3 generations in the apical endpoints of survival and reproduction. Employing a suite of acute and chronic bioassays, including chronic exposures spanning multiple generations, allows for a more robust screening‐level evaluation of the potential impact of chlorpyrifos on aquatic receptors for variable periods of exposure. Environ Toxicol Chem 2022;41:1054–1065. © 2021 SETAC