Wild aquatic birds are the major reservoir for influenza A viruses (FLUAV) and play a key role in viral maintenance and dissemination. Likewise, swine have a vital role in the adaptation, and reassortment of FLUAVs that may result in zoonotic strains of pandemic concern. Despite increased surveillance efforts, the diversity and ecology of FLUAVs circulating in wild birds and swine in Latin America remain understudied. For instance, the 2009 H1N1 influenza pandemic strain emerged in Mexico, but its circulation remained undetected in pigs before its detection in humans.The studies presented here aimed to better define the ecology and evolution of FLUAVs in wild aquatic birds and swine, using samples from an understudied region of the world: Guatemala, in Central America. I used targeted sequencing on the FLUAV genome from wild birds and swine swabs, and bioinformatics tools to analyze full genome sequencing data generated by Next-Generation Sequencing technologies to characterize the potential genetic traits of these viruses to cross the interspecies barrier. The first part of this thesis comprises the results of analyzing six migratory seasons (2013-2019) of avian FLUAV surveillance efforts in Guatemala, a stopover site for multiple overlapping flyways. Overall, I found great FLUAV diversity, in terms of subtype combinations and a high frequency of detection of viruses that are rarely found in breeding grounds in North America. My results provide additional support to previous observations that Guatemala can serve as a major geographic bottleneck with a significant role in the persistence and evolution of FLUAV in the region. The second part of this thesis describes the results of two years of FLUAV active surveillance in a commercial swine farm in Guatemala without a history of vaccination. We found a unique group of H1N1 pdm09 sequences that suggests independent evolution from similar viruses circulating in Central America. These viruses may represent the establishment of a novel genetic lineage with the potential to reassort with other co-circulating viruses, and whose zoonotic risk remains to be determined. My thesis has contributed to a better understanding of FLUAV ecology in Central America.