The communities making up Jefferson County were first founded just prior to the Revolutionary War. The area attracted would-be landowners seeking fertile soil and abundant sources of lumber and water.
In 1803, Hillsboro's first settlers, the Huskey family, settled the area to be close to Belew Creek, formed by three springs. Descendants of that family of farmers still live in the community and operate one of Missouri’s oldest farms.
Hillsboro and its neighboring communities developed quickly in the late 18th and early 19th centuries and, on December 8, 1818, Jefferson County was created. Herculaneum, located on Jefferson's far east border, was named the original county seat. By 1832, however, residents began calling for a more centralized county seat and received it when a stretch of land – later named Hillsboro – was donated by a local family.
After being named county seat, Hillsboro grew as a center of government. The first courthouse was completed in 1840 before being replaced by a larger and more sophisticated building in 1865. This second courthouse was modernized and expanded throughout its more than 100 years of use.
A community mostly consisting of government workers in its early years, Hillsboro grew in the years after being named county seat and was incorporated in 1873. A train station was built in the nearby community of Victoria in 1858, connecting the entire area to the rest of the continent. Rail travel was the primary mode of transportation for residents and visitors of the Hillsboro area until 1940, when Highway 21 was built. In 1939, the Hillsboro School District was established.