Despite the crucial role played by nautical cartography during the 16th-century Iberian Expansion, surviving examples of charts used at sea are extremely rare, leaving gaps in our knowledge of how they were used in practice during this pivotal period. The present paper sheds light on this matter by introducing one of earliest extant Portuguese charts presenting positional fixes, which have so far gone unnoticed by researchers: the anonymous chart of about 1524 kept at the James Ford Bell Library. It is demonstrated that a second chart, kept at the Harry Ransom Center, was made by the same author from the same pattern. The production context of the charts is evidenced through a description of the manuscript copying technique most likely employed, and the rules governing chart manufacture in 16th-century Portugal. Exceptional aspects of the charts’ placenames are discussed, and a possible source for their geographical design is identified.