Land Patent for Gray's Woodyard in Worcester County, Maryland dated October 10, 1743. The land was purchased near St. Martin's river by Joseph Gray from Joseph Scroggin and was located adjacent to James Stephen Bridell's plantation.

Worcester County was created by the division of the formerly larger Eastern Shore's Somerset County in 1742. The county seat, which was previously located near the confluence of Dividing Creek with the Pocomoke River, was later transferred to the river port of Snow Hill, at the head of navigation of the Pocomoke, now near the center of the new county.

Both the areas of Somerset and Worcester Counties were divided into old colonial divisions of "hundreds", from south to north: Mattapony, Pocomoke, Boquetenorton, Wicomico, and Baltimore Hundreds. Later subdivisions of these hundreds added Pitts Creek, Acquango, Queponco, and Buckingham & Worcester Hundreds, all of which in turn eventually became election districts for the newly independent state following American independence. Competing territorial claims between the Proprietor family of the Calverts and the Lords Baltimore in the old Province of Maryland and the Penns of the neighboring Province of Pennsylvania to the north and of what later became the state of Delaware to the east led to the surveying of Worcester County's northern border, the "Transpeninsular Line" in 1751, though boundary disputes continued through the rest of the colonial period, not totally settled until the work of the famous Charles Mason and Jeremiah Dixon with their "Mason–Dixon line". In 1779, Stephen Decatur, the famous United States Navy officer and hero of the First Barbary War and the Second Barbary War in the early 1800s, and leading into the War of 1812, was born at Sinepuxent, near what is today the town of Berlin.

Originally settled by European immigrants of British and Irish stock, along with bound laborers of mainly West African descent, Worcester County was divided during the colonial period into several Church of England parishes, though Quakers, Presbyterians, and later Methodists also set up meeting houses. Like the border states in general, Worcester County had a high proportion of free people of color for many decades before the Civil War, due in part to the influence of initially Quakerism, and later Methodism.

Worcester County was primarily an agricultural area from its inception, first planting tobacco, but when the quality produced in the area's sandy soil could not compete with that produced elsewhere, growing wheat, corn, and livestock. Early industrial activity included the smelting of bog iron ore in a brick blast furnace to make pig iron at Furnacetown in the first half of the 19th century. The presence of large bald cypress swamps along the Pocomoke River led to logging, the manufacture of roofing shingles, and shipbuilding along the river at Newtown (later Pocomoke City). The arrival of steam-powered water transport and then the railroad opened urban markets to another of Worcester County's principal products: seafood, particularly shellfish. Oysters, clams, and crabs were shipped to Baltimore, Philadelphia, and New York. Soon after the Civil War (to each side of which Worcester County sent soldiers), parts of both Worcester and Somerset Counties were combined to create, in 1867, Wicomico County. Also in the later 19th century, the seaside resort of Ocean City was founded.