We developed an in vitro micropropagation protocol for Agrimonia rostellata Wallr. (Rosaceae), a New York State–threatened native plant species. For initial experiments, we investigated methods for disinfection of nodal sections from field-grown plants before transfer to culture medium to generate contaminant-free plant material. Fungal contamination was most prevalent. The only effective treatment was a combination of washes in a detergent solution followed by treatment with 70% ethanol, 10% bleach, plus vacuum infiltration with a solution of the antifungal compound Miconazole. We also found it was necessary to include 20 mg/l Miconazole in the culture medium to prevent fungal contamination. A Murashige and Skoog–based medium supplemented with 0.1 mg/l indole-3-butyric acid (IBA), 5 g/l charcoal, and 7 g/l agar was the only medium tested that resulted in both the development of multiple shoots and rooting. A starting population of 35 contaminant-free, in vitro plants was scaled up to 1013 plants in 6 mo. All rooted plantlets were successfully established in a soilless mix and then transferred to the field. These methods proved to be effective for generating a large population of A. rostellata for replicated field studies to identify the cause(s) of its decline.