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Objectives: This study compared the prevalence of concentrated urine (urine specific gravity ≥1.021), an indicator of hypohydration, across Tsimane' hunter-forager-horticulturalists living in hot-humid lowland Bolivia and Daasanach agropastoralists living in hot-arid Northern Kenya. It tested the hypotheses that household water and food insecurity would be associated with higher odds of hypohydration., Methods: This study collected spot urine samples and corresponding weather data along with data on household water and food insecurity, demographics, and health characteristics among 266 Tsimane' households (N = 224 men, 235 women, 219 children) and 136 Daasanach households (N = 107 men, 120 women, 102 children)., Results: The prevalence of hypohydration among Tsimane' men (50.0%) and women (54.0%) was substantially higher (P < .001) than for Daasanach men (15.9%) and women (17.5%); the prevalence of hypohydration among Tsimane' (37.0%) and Daasanach (31.4%) children was not significantly different (P = .33). Multiple logistic regression models suggested positive but not statistically significant trends between household water insecurity and odds of hypohydration within populations, yet some significant joint effects of water and food insecurity were observed. Heat index (2°C) was associated with a 23% (95% confidence interval [CI]: 1.09-1.40, P = .001), 34% (95% CI: 1.18-1.53, P < .0005), and 23% (95% CI: 1.04-1.44, P = .01) higher odds of hypohydration among Tsimane' men, women, and children, respectively, and a 48% (95% CI: 1.02-2.15, P = .04) increase in the odds among Daasanach women. Lactation status was also associated with hypohydration among Tsimane' women (odds ratio = 3.35, 95% CI: 1.62-6.95, P = .001)., Conclusion: These results suggest that heat stress and reproductive status may have a greater impact on hydration status than water insecurity across diverse ecological contexts., (© 2020 Wiley Periodicals LLC.)