The present study details anatomical features of the lining of the emu and ostrich vagina, which in birds may impede the forward movement of spermatozoa. Vaginal samples were collected for light and electron microscopy. Samples for light microscopy were fixed in Bouin’s fluid, while samples for electron microscopy were fixed in 3% glutaraldehyde. After fixation the samples were processed routinely. A series of broad annular mucosal folds bearing convoluted primary folds were present in both ratites. The lining of the vaginal folds was a combination of ciliated, non-ciliated, vesicular and mitochondrial cells. The non-ciliated and a few ciliated cells, lining the crypts, contained mucin droplets. The role of the mucus is unclear. The results of the study show a similarity in the gross anatomical and scanning electron microscope features of the vagina in the emu and ostrich. Differences in the cellular composition of the vaginal epithelium were observed at the transmission electron microscope level.