Fletcher credited Frederic Ward Putnam for stimulating her interest in American Indian culture. She studied the remains of the Indian civilization in the Ohio and Mississippi valleys, became a member of the Archaeological Institute of America in 1879, and worked and lived with the Omahas as a representative of the Peabody Museum of Archaeology and Ethnology, Harvard University.
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These times marked the beginning of her 40-year association with an Omaha named Francis La Flesche. They collaborated professionally and also had an informal mother-son relationship. They lived together in Washington, D.C., beginning in 1890.
In 1883 she was appointed special agent to allot lands to the Miwok tribes, in 1884 prepared and sent to the World Cotton Centennial an exhibit showing the progress of civilization among the Indians of North America in the quarter-century previous, in 1886 visited the natives of Alaska and the Aleutian Islands on a mission from the commissioner of education, and in 1887 was United States special agent in the distribution of lands among the Winnebagoes and Nez Perces. She was made assistant in ethnology at the Peabody Museum in 1882, and received the Thaw fellowship in 1891 which was created for her. She was president of the Anthropological Society of Washington and of the American Folklore Society, and vice-president of the American Association for the Advancement of Science.
Working through the Womans National Indian Association, she introduced a system of making small loans to Indians, wherewith they might buy land and houses. Later she helped write, lobbied for and helped administer the Dawes Act of 1887 which broke up reservations and substituted individual ownership of land parcels.