Influenza A viruses initiate infection when the envelope glycoprotein hemagglutinin (HA) binds sialic?acid-containing receptors on host cells and mediates membrane fusion. HA?mediated cross?linking of erythrocytes forms the basis of the hemagglutination (HA) and hemagglutination?inhibition (HI) assays that are widely used to quantify virus and virus?specific antibodies. The choice of erythrocyte species significantly affects these assays, but data for large ratites are limited. This study compared the hemagglutination characteristics of two highly pathogenic avian influenza A (H5N1) isolates, A/H5N1/Bogor 2 and A/H5N1/Lawang, using erythrocytes from ostrich, emu, quail, chicken and horse. Blood was collected from clinically healthy adult animals (n = 5 per species) (wing vein for chickens and quails; jugular vein for ostriches, emus and horses). Washed erythrocytes were examined by bright?field microscopy and used in a microtiter HA assay in which serial two?fold dilutions of erythrocytes were mixed with a constant dose of virus (105 TCID50 per well). Hemagglutination patterns were recorded up to 300 min and expressed as the highest erythrocyte dilution showing complete agglutination. Ostrich and emu erythrocytes were markedly larger than those of chicken and quail, whereas horse erythrocytes were small and anucleate. All five species supported hemagglutination by both A(H5N1) isolates. For A/H5N1/Bogor 2, horse erythrocytes showed delayed agglutination and a subsequent decline in HA titer, whereas avian erythrocytes yielded high, stable titers. For A/H5N1/Lawang, HA titers were more homogeneous and largely time?independent across species. Overall, differences between virus strains were greater than those between erythrocyte species, suggesting that viral HA properties dominate over gross erythrocyte morphology in determining hemagglutination and that the optimal erythrocyte species for HA and HI testing of H5N1 viruses may vary among strains.