<p>Domestic horses re-entered North America in the 16th century, spreading from Spanish colonies in Mexico into the central Great Plains before the late 17th century. The routes of their dispersal to the Northern Plains are less apparent and no historic horses have been genetically studied from this grassland region. We analyzed a horse cheek tooth found in the Little Missouri River valley, North Dakota, performing mitochondrial genome sequencing, radiocarbon dating and sequential analysis of δ¹³C and δ¹⁸O values in the dental enamel. Radiocarbon dating indicated this horse most likely lived in the late 18th century (1730–1807&#xa0;cal CE). The horse mtDNA sequence belonged to Haplogroup L but has not previously been reported in modern and historic horses. Further, it exclusively clustered with mtDNA haplotypes from Thoroughbred horses of British origin representing the first report about the relationship between the historic North American and British horses. Serial analysis of δ¹³C and δ¹⁸O values revealed that the tooth belonged to a horse that consumed C3 plants representing the main vegetative component of the region with distinctive cold and warm seasons and supported the horse residence in the Northern Plains for the duration of its life.</p>