The area which now comprises Karnes County has been the site of human habitation for several millennia. Archeological evidence reveals that hunter-gatherer Indians of the Coahuiltecan linguistic family occupied the region for several thousand years prior to the arrival of Europeans. It was also in the hunting range of Comanche, Tonkawa, Karankawa, and Lipan Apache Indians. The region was also inhabited by the Pataguilla Indians, who lived in the San Antonio River valley between the sites of present-day Panna Maria and Falls City, and the Pitaias Indians, who lived near the site of present Conquista Crossing.

Around 1770, the region became the nucleus of ranching activity between San Antonio de Béxar and La Bahía (now Goliad). The Spanish established a fort called Fuerte de Santa Cruz del Cíbolo on Cibolo Creek near the site of present Czestochowa to protect the ranches in the area from raids by Comanches and other Indian tribes. In 1783, after repeated Comanche attacks, the fort and some twenty-five neighboring ranches were abandoned, and by the mid-1780s only six ranches and eighty-five Spanish settlers remained.

The original Hernández and Menchaca ranches were divided up by heirs of the families, and some of the land sold to other families, including the Veramendi, Cassiano, Flores, Navarro, and Carillo clans. By the 1840s the first Anglo-American settlers began arriving in the region. The first Anglo-American settlement in the county was made in 1852 at Helena at the site of an earlier Mexican settlement called Alamita.

By 1853 Anglo settlers, led by Thomas Ruckman and Lewis S. Owings who had founded Helena, petitioned the state legislature to form a new county from portions of Bexar, Gonzales, DeWitt, Goliad, and San Patricio counties. On February 4, 1854, the legislature complied, passing a measure to establish a new county, named Karnes for Texas revolutionary leader Henry Wax Karnes, with Helena as county seat.

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