Among prehistoric native groups of North America, the Ohio Hopewell was a particularly notable cultural manifestation that prevailed during the Middle Woodland Period (ca. 2100 -1500 BP). The group is defined by the construction of elaborate burial mounds, evidence of ritual burning and cremation ceremonies, The presence of anthraquinones in these Ohio Hopewell textiles, and the absence of the bloodroot alkaloids, supports previous studies suggesting the prehistoric use of Galium species as dye plants in Eastern North America.Statement of significance Among the prehistoric native groups of North America, the Hopewell cultural manifestation are particularly notable for their elaborate ceremonial mounds, and the intricately decorated objects they made. While few textiles have survived, those fragments which remain provide important evidence for the technological knowledge and craftsmanship of the people of the past. A rapid method that requires a very small sample yields information about the red dye colorants employed in this fabrics. Direct analysis in real time-time-of-flight mass spectrometry (DART-MS) was used to examine fibers from red-and yellow-coloured textiles from the Seip Mound Group in Ohio, USA. The analysis showed that compounds characteristic of bedstraw plants of the Galium genus, rather than those from bloodroot, provide the red colour.This supports previous work suggesting the use of bedstraw for dyeing in prehistoric Eastern North America.