This study was performed to quantify the association between mortality and known and unknown secondhand smoke (SHS) exposure as measured by cotinine levels in non-smokers. Data collected from 1999 to 2010 in the National Health and Nutrition Examination Survey (NHANES) were linked to the National Death Index. Self-reported non-smokers aged ≥20 years (N = 20 175) were studied. Serum cotinine was measured at recruitment; non-smokers were those with cotinine below the reported race-specific cut-off points (5.92, 4.85 and 0.84ng/ml for non-Hispanic blacks, non-Hispanic whites and Mexican Americans, and 3.08ng/ml for all other groups). Serum cotinine levels were significantly associated with overall survival (HRadj 1.17, 95% CI: 1.13–1.22 per natural-log unit change in cotinine), death for all medical causes, lung cancer, all cancers and heart diseases, after adjustment for gender, race/ethnicity, body mass index, smoking history and education. Similar results were observed when non-smokers reporting no SHS exposure at home or work were analyzed. There was a statistically significant trend in years of life lost (YLL), adjusted for confounders, across cotinine categories both in non-smokers (YLLadj: 5.6, 6.4, 6.8, 7.5; P for trend < 0.0001) and non-smokers reporting no SHS exposure (YLL adj: 5.5, 6.1, 6.3, 6.7; P for trend = 0.002). Serum cotinine levels identify SHS-attributable mortality in subjects who would have otherwise been overlooked by questionnaire data, providing further evidence that the economic toll of SHS may be substantially higher than what was reported based on questionnaires.