16Receptor recognition and binding is the first step of viral infection and a key determinant of host 17 specificity. The inability of avian influenza viruses to effectively bind human-like sialylated receptors 18 is a major impediment to their efficient transmission in humans and pandemic capacity. Influenza 19H9N2 viruses are endemic in poultry across Asia and parts of Africa where they occasionally infect 20 humans and are therefore considered viruses with zoonotic potential. We previously described 21 H9N2 viruses, including several isolated from human zoonotic cases, showing a preference for 22human-like receptors. Here we take a mutagenesis approach, making viruses with single or multiple 23 substitutions in H9 haemagglutinin to determine the genetic basis of preferences for alternative 24 avian receptors and for human-like receptors. We describe amino acid motifs at positions 190, 226 25and 227 that play a major role in determining receptor specificity, and several other residues such 26as 159, 188, 193, 196, 198 and 225 play a smaller role. Furthermore, we show changes at residues 27 135, 137, 147, 157, 158, 184, 188, and 192 can also modulate virus receptor avidity and that 28 substitutions that increased or decreased the net positive charge around the haemagglutinin 29 receptor-binding site show increases and decreases in avidity, respectively. The motifs we identify 30 as increasing preference for the human-receptor will help guide future H9N2 surveillance efforts 31 and facilitate our understanding of the emergence of influenza viruses with high zoonotic potential. 32