Short-term exposure to low temperatures can lower pollen viability and germination in strawberry. Fruit malformation was found to be negatively correlated with pollen germination. Ultrastructural analysis was performed at various developmental stages of anthers and pollen grains of the strawberry upon exposure to short-term low temperature (STLT). ‘Maehyang’ strawberry was exposed to 2 oC from 6 pm to 9 am for 3 days during different flower developments before anthesis, whereas the plants in control remained in the greenhouse. The epidermis did not develop well and was deformed after STLT. The epidermis showed a significantly lower cell expansion index in the short-term low-temperature treatment than in control at the vacuolated microspore, bicellular pollen, and mature pollen stages, indicating significant deformation of the epidermal cells by low temperature. The projected area of starch was significantly greater in STLT treatment than in control at the mature pollen stage. The relative fluorescence intensity of anthers was significantly higher in STLT treatment than in control at the sporogenous cell and microspore mother cell stages, indicating that low temperature substantially inhibited anther development at early stages. The results showed that STLT stress caused abnormal development of the ultrastructure of the anther wall and pollen grains, along with starch over-accumulation during pollen development. These abnormal developments may lead to poor pollen germination and thus cause fruit malformation in strawberry.