<p><i>Bertholletia excelsa</i> (Lecythidaceae), commonly known as the Brazil nut, stands as a pivotal species in sustainable rainforest management, offering a multinational solution for long-term rainforest stewardship in the Amazon. Despite its economic and ecological significance, fundamental genetic data such as genome size and detailed chromosomal analysis still need to be included. Here, we present the first report on the genome size estimation of <i>B. excelsa</i>, determined through flow cytometry analysis to be 654.87 Mbp, with a confirmed chromosome count of 2n = 34. A tentative classification of chromosome morphology reveals a predominance of submetacentric chromosomes, indicating a potential karyotypic asymmetry. Furthermore, we provide insights into chromosome size estimation in Mbp, serving as a crucial reference for future genome assembly efforts. Notably, <i>B. excelsa</i> becomes only the second species within the Lecythidaceae family with a characterized genome size. Our findings underscore the wide variation in genome sizes within the family, emphasizing the importance of comprehensive genetic characterization efforts for neotropical and tropical species. This study contributes with genetic data essential for the next steps towards the genome-wide studies with positive potential implications for the conservation and sustainable management of <i>B. excelsa</i> and highlights avenues for further research in rainforest biodiversity conservation.</p>