Abstract Florida Cracker sheep is a heritage sheep breed and an endangered species that was developed under natural selection in Florida, US. The objective of this study was to evaluate the effect of short-term heat stress on the plasma metabolome of Florida Cracker ewes (2 yr). A total of fourteen ewes were allocated to heat stress (HT, n = 7) or cooling (CTL, n = 7) conditions for 6 wk during the summer season. Treatment groups were grouped in covered concrete floor pens with similar dimensions and characteristics and had access to shelter, feed, and freshwater ad libitum. The HT pen was exposed to regular ambient temperatures, and for the cooling pen, an evaporative cooling fan was used. Data for ambient temperature and relative humidity of HT and CTL pens were recorded every week to estimate the temperature humidity index (THI). Blood samples were collected from each experimental ewe 1 wk before the start of the experiment (baseline) and at wk 1, 2, 3, 4, 5, and 6 post-treatment. Plasma was extracted from the blood and processed for targeted metabolomics. Targeted metabolomics was performed using Nuclear Magnetic Resonance spectroscopy to quantify a total of 50 metabolites, including organic acids, amino acids, hexoses, lipids, and carnitines. Statistical analysis for biomarker and pathway enrichment was performed using Metaboanalyst 5.0. The THI values for the HT and THI groups ranged from 81.3 to 89.5 and from 73.5 to 75.6, respectively. A total of 19 differentially abundant metabolites were identified between HT and CTL. The pathway enrichment analysis showed upregulation of fatty acid and primary bile biosynthesis, and glutathione, porphyrin, glycine, serine, and threonine metabolism (P ≤ 0.05) in HT compared with CTL sheep. These metabolic pathways could have a role in the response of the ewes to short-term HT. Our findings highlight that short-term HT alters the plasma metabolome of Florida Cracker ewes. Understanding these changes can contribute to the development of effective management strategies to mitigate the detrimental impact of HT on sheep operations in the Southern US.