While hair samples are easier to collect and less expensive to store and transport than biological fluids, and hair nicotine characterizes tobacco exposure over a longer time period than blood or urine cotinine, information on its utility, compared with salivary cotinine, is still limited. We conducted a cross-sectional study with 289 participants (107 active smokers, 105 passive smokers with self-reported secondhand smoke (SHS) exposure, and 77 non-smokers with no SHS exposure) in Baltimore (Maryland, USA). A subset of the study participants (n = 52) were followed longitudinally over a two-month interval. Median baseline hair nicotine concentrations for active, passive and non-smokers were 16.2, 0.36, and 0.23 ng/mg, respectively, while those for salivary cotinine were 181.0, 0.27, and 0.27 ng/mL, respectively. Hair nicotine concentrations for 10% of passive or non-smokers were higher than the 25th percentile value for active smokers while all corresponding salivary cotinine concentrations for them were lower than the value for active smokers. This study showed that hair nicotine concentration values could be used to distinguish active or heavy passive adult smokers from non-SHS exposed non-smokers. Our results indicate that hair nicotine is a useful biomarker for the assessment of long-term exposure to tobacco smoke.