For an efficient harvest of self-fertilized seeds in spinach, the effect of temperature on the expression of gynomonoecy was studied by employing selfed-seed populations from three gynomonoecious plants that produced 525, 359 and 186 seeds/plant. These selfed-seed populations were named P-1, P-2, and P-3, respectively. Seeds, sown monthly from December to March in an unheated glasshouse, revealed that the frequency of gynomonoecious plants was highest in P-1 and lowest in P-3 at any sowing time. In December-sown seeds of P-1, the frequency of gynomonoecious plants was 33%; as the sowing time was delayed, the frequency of gynomonoecious plants increased until it reached 100% in the March-sown seeds. Similar results were obtained in a growth chamber kept at 15, 20 and 25°C, and under 16 h photoperiod with 120 μmol · m⁻² · s⁻¹ PPFD. However, the frequency of hermaphroditic flowers on gynomonoecious plants was affected by temperature in all three populations; it was lowest at 15°C and highest at 20°C. At 25°C, hermaphroditic flowers occurred near the shoot apex, but the number of nodes with hermaphroditic flowers was equal or less than at 20°C due to the delay in bolting and/or reduced plant height. When seedlings were transferred to 25°C after bolting was induced at 20°C for 18 days, the mean plant height was reduced but the number of nodes with hermaphroditic flowers was unaffected. This cultural manipulation seems to be advantageous for seed production because it suggests that a line with a strong expression of gynomonoecy can be selected to maximize the production of self-fertilized spinach seeds.