Aerenchyma, a gaseous space in shoots and roots, is critical for plants to adapt and survive under environmental stresses, such as hypoxia, drought, and nutritional deficiency. Although ethylene-mediated signaling cascades, including oxidative burst and Ca2+ accumulation, have been elucidated to regulate aerenchyma formation, the specific mechanisms remain unclear, and our understanding of the developmental stages and structural characteristics of aerenchyma is still very limited. We analyzed the aerenchyma formation process in two species of duckweeds, Spirodela polyrhiza (S. polyrhiza) and Lemna minor (L. minor), which are free-floating aquatic plants with highly developed aerenchyma, applying X-ray micro-computed tomography to reveal the three-dimensional structure of aerenchyma. Aerenchyma development in duckweeds was closely linked with guard cell development, as guard cells developed, new cavities were created beneath mature guard cells and the connectivity of individual intracellular spaces increased. This process was accompanied by programmed cell death, which was utilized differently in spatiotemporal contexts in the two species. Further investigations using developmental stage- and layer-specific transcriptome analysis combined with pharmacological studies revealed that spatiotemporal balance of phytohormones including ethylene, abscisic acid and, salicylic acid plays important roles in the development and structural patterning of aerenchyma. Moreover, transcriptome analysis unveiled notable structural disparities between the adaxial and abaxial surfaces of S. polyrhiza, highlighting cellular-level adaptations to their aquatic environment. These novel insights significantly advance our understanding of aerenchyma formation and its regulatory pathways across diverse plant taxa.