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Uta stansburiana are an emerging model system for studying sexual selection, polymorphism, and the evolution of pace-of-life syndromes (POLS) whose distribution covers variable environments and a wide latitudinal gradient. POLS are suites of traits causing variation of life history along a slow maturing-fast maturing continuum. We present a high-quality chromosome-level reference genome for U. stansburiana and pair it with RNA-seq gene expression data to demonstrate, for the first time, the molecular basis for pace-of-life differences between locations with higher and lower climate seasonality and sexual size dimorphism (SSD). Our assembly is 2.1 Gbp, has scaffold N50 of 320 Mbp, includes 104 scaffolds, and has an L50 of 3. The assembly comprises six macrochromosomes and 11 microchromosomes. We annotated 20,350 genes for the assembly and found a repeat element composition of 49.23%, similar to work in other phrynosomatid lizards. RNA-seq gene expression data demonstrate expression differences in genes associated with pace-of-life differences including those related to stress, sexual reproduction, and cell proliferation/carcinogenesis between different environments. Our results provide the first differential gene expression evidence of environmentally-mediated pace-of-life processes related to different degrees of SSD in U. stansburiana and demonstrate the utility of RNA-seq gene expression data in detecting POLS.