From 1863 to 1868 Gilbert worked for the Ward Natural Science Establishment, a firm that manufactured and distributed scientific equipment for schools. In 1869 he joined the second Ohio State Geological Survey as a volunteer assistant and in 1871 was assigned to the George M. Wheeler survey west of the 100th meridian. During his three-year service with the survey, he made a remarkable journey by boat up the lower canyons of the Colorado River, by pack train through central Arizona and down the valley of the Gila River, and again by boat down the Colorado to the Gulf of California. During that trip he made observations and gathered evidence that the post-Carboniferous (less than 280,000,000 years old) strata of Europe and eastern North America did not extend throughout the world. He published two papers characterizing the Basin and Range and Plateau provinces and naming and describing the Pleistocene Lake Bonneville, which was the ancestor of the Great Salt Lake in Utah.

In 1875 Gilbert was transferred to the John Wesley Powell survey, which took him to Utah. When the U.S. Geological Survey was formed in 1879, he was made one of the six senior geologists. In 1884 he was placed in charge of the Appalachian division of geology, and in 1889, upon the creation of the division of geologic correlation, he was placed at its head. After 1892 he relinquished his position as chief geologist and most of his administrative duties in order to return to deeper study of some of the problems he had investigated earlier. The Bonneville Monograph (1890) was regarded as his magnum opus. His Report on the Geology of the Henry Mountains (1877), in which the intrusive igneous structure known as a laccolith was first described, and his History of the Niagara River (1890) were of particular importance. He also played an important part in the planning of the U.S. Geological Survey's bibliographic work and the adoption of the principles of nomenclature and cartography that form the basis of the survey's geological map work.