Highly pathogenic avian influenza (HPAI) A/H5N1, clade 2.3.4.4b, has spread globally, with the first outbreak in a commercial poultry farm in Brazil reported in 2025. This study aimed to genetically characterize A/H5N1 samples from multiple Brazilian regions using whole-genome next-generation sequencing (NGS) to investigate viral diversity and reassortment events. Phylogenetic analyses revealed that Brazilian isolates segregated into two distinct clades: one showing high similarity to viruses detected in Argentina in 2025 suggesting adaptation of new reassortants along a regional dissemination route in South America; and another clustering with North American isolates, indicating an independent introduction. Genotyping confirmed the emergence of previously unreported genomic rearrangements in South America, underscoring the complexity of viral evolution in the region. These findings highlight intra-epidemic genomic diversity and reinforce the need for continuous, in-depth molecular surveillance to monitor the dynamics and potential impacts of A/H5N1 on animal and public health.