It is from the American Revolution:

“In the fall 1775, the U.S. Navy established to intercept British ships carrying war supplies to their trops in the colonies. The second continental congress authorized the mustering of five Marine Companies to accompany be Navy on their first mission. The first Marines that enlisted when from Philadelphia, and they carried drums painted yellow, depicting a coiled rattlesnake with thirteen rattles, and the motto "Don't Tread On Me." At the Congress, Christopher Gadsden of South carolina served on the marine committee that was outfitting the first naval mission. Gasden presented the newly appointed commander in chief of the Navy, commodore Esek Hopkins, the yellow rattlesnake flag to serve as his distinctive personal standard. "Don't Tread On Me" and the rattlesnake depicted are two symbols widely associated with the American Revolution and independence.”