This study identifies an influenza A virus from little yellow-shouldered bats in Guatemala, which is significantly divergent from known influenza A viruses. The virus, designated as "A/little yellow-shouldered bat/Guatemala/164/2009" (A/bat/Guat/09), has a hemagglutinin (HA) gene that diverged at a similar time to known subtypes, leading to the classification of a new subtype, H17. The neuraminidase (NA) gene is highly divergent, and the internal genes diverged before the divergence of known influenza A lineages. The virus was unable to be propagated in cell cultures or chicken embryos but showed compatibility with human influenza viruses in human cells, suggesting potential for reassortment and the generation of new pandemic or panzootic influenza A viruses. This discovery expands the known reservoirs of influenza A viruses and highlights the need for further research on the maintenance and evolution of these viruses in bat populations.This study identifies an influenza A virus from little yellow-shouldered bats in Guatemala, which is significantly divergent from known influenza A viruses. The virus, designated as "A/little yellow-shouldered bat/Guatemala/164/2009" (A/bat/Guat/09), has a hemagglutinin (HA) gene that diverged at a similar time to known subtypes, leading to the classification of a new subtype, H17. The neuraminidase (NA) gene is highly divergent, and the internal genes diverged before the divergence of known influenza A lineages. The virus was unable to be propagated in cell cultures or chicken embryos but showed compatibility with human influenza viruses in human cells, suggesting potential for reassortment and the generation of new pandemic or panzootic influenza A viruses. This discovery expands the known reservoirs of influenza A viruses and highlights the need for further research on the maintenance and evolution of these viruses in bat populations.