Background:Extreme heat is a leading weather-related cause of mortality in the United States, but little guidance is available regarding how temperature variable selection impacts heat–mortality relationships.Objectives:We examined how the strength of the relationship between daily heat-related mortality and temperature varies as a function of temperature observation time, lag, and calculation method.Methods:Long time series of daily mortality counts and hourly temperature for seven U.S. cities with different climates were examined using a generalized additive model. The temperature effect was modeled separately for each hour of the day (with up to 3-day lags) along with different methods of calculating daily maximum, minimum, and mean temperature. We estimated the temperature effect on mortality for each variable by comparing the 99th versus 85th temperature percentiles, as determined from the annual time series.Results:In three northern cities (Boston, MA; Philadelphia, PA; and Seattle, WA) that appeared to have the greatest sensitivity to heat, hourly estimates were consistent with a diurnal pattern in the heat-mortality response, with strongest associations for afternoon or maximum temperature at lag 0 (day of death) or afternoon and evening of lag 1 (day before death). In warmer, southern cities, stronger associations were found with morning temperatures, but overall the relationships were weaker. The strongest temperature–mortality relationships were associated with maximum temperature, although mean temperature results were comparable.Conclusions:There were systematic and substantial differences in the association between temperature and mortality based on the time and type of temperature observation. Because the strongest hourly temperature–mortality relationships were not always found at times typically associated with daily maximum temperatures, temperature variables should be selected independently for each study location. In general, heat-mortality was more closely coupled to afternoon and maximum temperatures in most cities we examined, particularly those typically prone to heat-related mortality.Citation:Davis RE, Hondula DM, Patel AP. 2016. Temperature observation time and type influence estimates of heat-related mortality in seven U.S. cities. Environ Health Perspect 124:795–804; http://dx.doi.org/10.1289/ehp.1509946