The urban heat island (UHI) is a well-documented effect of urbanization on local climate, identified by higher temperatures compared to surrounding areas, especially at night and during the warm season. The details of a UHI are city-specific, and microclimates may even exist within a given city. Thus, investigating the spatiotemporal variability of a city’s UHI is an ongoing and critical research need. We deploy ten weather stations across Knoxville, Tennessee, to analyze the city’s UHI and its differential impacts across urban neighborhoods: two each in four neighborhoods, one in more dense tree cover and one in less dense tree cover, and one each in downtown Knoxville and Ijams Nature Center that serve as control locations. Three months of temperature data (beginning 2 July 2014) are analyzed using paired-sample t tests and a three-way analysis of variance. Major findings include the following: (1) Within a given neighborhood, tree cover helps negate daytime heat (resulting in up to 1.19 ∘C lower maximum temperature), but does not have as large of an influence on minimum temperature; (2) largest temperature differences between neighborhoods occur during the day (0.38–1.16 ∘C difference), but larger differences between neighborhoods and the downtown control occur at night (1.04–1.88 ∘C difference); (3) presiding weather (i.e., air mass type) has a significant, consistent impact on the temperature in a given city, and lacks the differential impacts found at a larger-scale in previous studies; (4) distance from city center does not impact temperature as much as land use factors. This is a preliminary step towards informing local planning with a scientific understanding of how mitigation strategies may help minimize the UHI and reduce the effects of extreme weather on public health and well-being.