Thalictrum cooleyi (Cooley's meadowrue, Ranunculaceae) is a federally endangered dioecious herb of wet pine savannas of the southeastern United States. We studied aspects of T. cooleyi biology crucial to conservation including its association with woody species, reproduction, and genetic structure in 11 populations in North Carolina and Georgia. Thalictrum cooleyi presence was negatively associated with canopy closure, but positively associated with shrub cover, including wax myrtle Morella cerifera. Deviations from strict dioecy are uncommon in T. cooleyi. Sex ratios differed significantly from 1:1, but despite male-bias in populations, pollen supplementation experiments found evidence for pollen limitation to seed set. Seeds are dormant at maturity. Cold stratification or gibberellic acid can break dormancy. Warm temperatures followed by cold stratification may increase germinability. Germination rates were low, likely due to low seed viability. Thalictrum cooleyi can be propagated vegetatively from caudex and rhizome divisions. AFLP analyses suggested that genetic diversity within and among populations is low. North Carolina and Georgia populations are genetically distinct, yet these limited data do not support hypotheses of separate species status or hybrid swarms. Our work suggests that habitat loss (i.e., transition of open pine savanna to dense forest), pollen limitation of seed set, poor seed viability, and possibly low genetic diversity within populations are major challenges to the recovery of T. cooleyi.