Cotyledons, food reserve organs, have been routinely used as explants in sunflower (Helianthus annuus L.) tissue culture, but the underlying mechanism is not well understood. The objective of this study was to investigate the changes of the cytological characteristics and chemical content of cotyledon explants under in vitro culture and their relationship with shoot regeneration ability. Cotyledons of 0-, 1-, and 7-day old germinating seeds of the F1 hybrid (Pacific 22 × Prado Red) and Prado Red varieties were placed with the adaxial side on a modified MS medium for 21 days, then subjected to cytological and food reserve composition analysis. The results showed that there was a statistically significant difference among cotyledon ages and conditions in terms of cytological features and food reserve composition. The cell numbers per area were lower in cotyledons cultured in vitro compared to those in germinating seeds, whereas the cell area and cotyledon thickness exhibited opposite results. The in vitro cotyledons showed lower contents of total fats, total sugar, and sucrose, but a higher content of protein. Correlation analysis demonstrated a positive relationship between shoot regeneration with the protein and cell area, but a negative relationship with cell numbers, indicating that high protein content and cell expansion may contribute to shoot regeneration ability in sunflowers. © 2019, Suranaree J. Sci. Technol.