The highly pathogenic Avian Influenza virus (HPAIV) H5N1 has caused a global outbreak affecting both wild and domestic animals, predominantly avian species. To date, cases of the HPAIV H5 Clade 2.3.4.4b in penguins have exclusively been reported in African Penguins. In Chile, the virus was confirmed in pelicans in December 2022 and subsequently spread across the country, affecting several species, including Humboldt penguins. This study aims to provide an overview of the incidents involving stranded and deceased Humboldt penguins and establish a connection between these events and HPAIV H5N1. Historical data about strandings between 2009 and 2023 was collected, and samples from suspected cases in 2023 were obtained to confirm the presence of HPAIV H5N1. Between January and August 2023, 2,788 cases of stranded and deceased penguins were recorded. Out of these, a total of 2,712 penguins deceased, evidencing a significative increase in mortality starting in early 2023 coinciding with the introduction and spreading of HPAIV H5N1 in the country. Thirty-seven events were categorized as mass mortality events, with the number of deceased penguins varying from 11 to 98. Most cases (97 %) were observed in the North of Chile. One hundred and eighty-one specimens were subjected to HPAIV diagnosis, four of which tested positive for HPAIV H5N1. Spatial analysis validates the correlation between mass mortality events and outbreaks of HPAIV in Chile. However, the limited rate of HPAIV H5N1 detection, which can be attributed to the type and quality of the samples, requiring further exploration.