The organotypic human air-liquid-interface (ALI) airway tissue model has been used as an in vitro cell culture system for evaluating the toxicity of inhaled substances. ALI airway cultures are highly differentiated, which has made it challenging to evaluate genetic toxicology endpoints. In the current study, we assayed DNA damage with the high-throughput CometChip assay and quantified mutagenesis with Duplex Sequencing, an error-corrected next-generation sequencing method capable of detecting a single mutation per 107 base pairs. Fully differentiated human ALI airway cultures were treated from the basolateral side with 6.25 to 100 μg/mL ethyl methanesulfonate (EMS) over a period of 28 days. CometChip assays were conducted after 3 and 28 days of treatment, and Duplex Sequencing after 28 days of treatment. Treating the airway cultures with EMS resulted in time- and concentration-dependent increases in DNA damage and a concentration-dependent increase in mutant frequency. The mutations observed in the EMS-treated cultures were predominantly C → T transitions and exhibited a unique trinucleotide signature relative to the negative control. Measurement of physiological endpoints indicated that the EMS treatments had no effect on anti-p63-positive basal cell frequency, but produced concentration-responsive increases in cytotoxicity and perturbations in cell morphology, along with concentration-responsive decreases in culture viability, goblet cell and anti-Ki67-positive proliferating cell frequency, cilia beating frequency, and mucin secretion. The results indicate that a unified 28-day study can be used to measure several important safety endpoints in physiologically relevant human in vitro ALI airway cultures, including DNA damage, mutagenicity, and tissue-specific general toxicity.