Hookah smoking has become common in the USA, especially among young adults. This study measured biomarkers of exposure to known tobacco product toxicants in a population-based sample of exclusive, established hookah users. Urinary biomarker data from 1753 adults in Wave 1 of the Population Assessment of Tobacco and Health (PATH) Study were used to compare geometric mean concentrations of biomarkers of exposure in exclusive, established past 30-day hookah users to never users of tobacco. Geometric mean ratios were calculated comparing hookah user groups with never users adjusting for age, sex, race/ethnicity, education, past 30-day marijuana use, secondhand smoke exposure and creatinine. Past 30-day hookah users (n = 98) had 10.6 times the urinary cotinine level of never tobacco users. Compared to never tobacco users, past 30-day hookah users had 2.3 times the level of the carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a metabolite of the tobacco-specific nitrosamine (TSNA) 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 1.3 times higher polycyclic aromatic hydrocarbons (PAHs) 3-hydroxyfluorene and 1-hydroxypyrene, 1.8 times higher levels of acrylonitrile, 1.3 times higher levels of acrylamide, and 1.2 times higher levels of acrolein exposure. These data indicate that hookah use is a significant source of exposure to nicotine, carcinogens, and respiratory toxicants.