Previous field and experimental studies have demonstrated that heterosubtypic immunity (HSI) is a potential driver of Influenza A virus (IAV) prevalence and subtype diversity in mallards. Prior infection with IAV can reduce viral shedding during subsequent reinfection with IAV that have genetically related hemagglutinins (HA). In this experiment, we evaluated the effect of HSI conferred by an H3N8 IAV infection against increasing challenge doses of closely (H4N6) and distantly (H6N2) related IAV subtypes in mallards. Two groups of thirty 1-month-old mallards each, were inoculated with 105.9 50% embryo infectious doses (EID50) of an H3N8 virus or a mock-inoculum. One month later, groups of five birds each were challenged with increasing doses of H4N6 or H6N2 virus; age-matched, single infection control ducks were included for all challenges. Results demonstrate that naïve birds were infected after inoculation with 103 and 104 EID50 doses of the H4N6 or H6N2 virus, but not with 102 EID50 doses of either IAV. In contrast, with birds previously infected with H3N8 IAV, only one duck challenged with 104 EID50 of H4N6 IAV was shedding viral RNA at 2 days post-inoculation, and with H6N2 IAV, only birds challenged with the 104 EID50 dose were positive to virus isolation. Viral shedding in ducks infected with H6N2 IAV was reduced on days 2 and 3 post-inoculation compared to control birds. To explain the differences in the dose necessary to produce infection among H3-primed ducks challenged with H4N6 or H6N2 IAV, we mapped the amino acid sequence changes between H3 and H4 or H6 HA on predicted three-dimensional structures. Most of the sequence differences occurred between H3 and H6 at antigenic sites A, B, and D of the HA1 region. These findings demonstrate that the infectious dose necessary to infect mallards with IAV can increase as a result of HSI and that this effect is most pronounced when the HA of the viruses are genetically related.