Genetic research on influenza virus biology has been informed in large part by nucleotide variants present in seasonal or pandemic samples, or individual mutants generated in the laboratory, leaving a substantial part of the genome uncharacterized. Here, we have developed a single-nucleotide resolution genetic approach to interrogate the fitness effect of point mutations in 98% of the amino acid positions in the influenza A virus hemagglutinin (HA) gene. Our HA fitness map provides a reference to identify indispensable regions to aid in drug and vaccine design as targeting these regions will increase the genetic barrier for the emergence of escape mutations. This study offers a new platform for studying genome dynamics, structure-function relationships, virus-host interactions, and can further rational drug and vaccine design. Our approach can also be applied to any virus that can be genetically manipulated.