A morphological study of female genital papillae of Etheostoma was conducted to examine the evolution of papillae as predicted by molecular phylogenies, and to examine correlations between papilla morphology and spawning behaviors and oviposition. Thirteen characters were used to describe variation in papillae in 128 species. Based on maximum likelihood ancestral state reconstructions, we corroborated several molecular phylogenetic hypotheses with morphology and developed hypotheses on several functional traits/adaptive traits and their associations with spawning behaviors. Papillar synapomorphies supported a close relationship between Allohistium and (Etheostoma Nanostoma) Ulocentra, a monophyletic Catonotus, and a sister-group relationship between E. vitreum and Boleosoma. Results suggest that the most recent common ancestor of Etheostoma buried eggs and had a simple tube with a distal, posteriorly oriented genital pore. All egg-burying species, except species of Nothonotus, have tube papillae that inject eggs into substrate. Females of Nothonotus have short, mound, and mound-tube papillae and typically bury their entire body to deposit eggs. Egg-clumping species have mound papillae. Most species that attach eggs to objects above the substrate, the most common behavior in Etheostoma, have tube papillae; the only exceptions are E. (Psychromaster) pallididorsum with a mound papilla, and E. (Vaillantia) davisoni with a mound-tube papilla. All egg-clustering species have complex, wide, and pleated rosette papillae with ventrally directed genital pores for adpressing eggs to the undersides of rocks or logs. The only species with a rosette papilla that is not an egg-clusterer is E. vitreum, which has been described as a communal egg-attacher. The papilla of E. vitreum is unique in being rosette with dense villi and a bifurcate papillar platform. The basal platform, swollen tissue immediately posterior to the base of the genital papilla that probably enhances maneuverability and precision of oviposition, appeared at the base of a large clade including all egg-clustering species and a large number of egg-attaching species. Adaptations in papilla morphology likely played a role in the expanded use of stream habitats and the diversification of darters.