For hybrid seed production, it is prerequisite to know the floral biology of the crop and pollen biology is an integral part of flower biology of any crop to understand artificial hybridization more efficiently. After pollination, germination of the viable pollen is important for proper fertilization of ovary and to develop optimum fruit and seed set. So, the study aimed to evaluate the in vitro pollen germination and viability of brinjal in ambient as well as cold storage conditions. The pollen germination was tested in vitro, by incubating the pollen grains for 3 hours at 25ᵒ C in different concentrations of sucrose, boron and Agar media [(T1 = 2 % Sucrose + 4 ppm Boron, T2 = 2 % Sucrose + 8 ppm Boron, T3 = 5 % Sucrose + 4 ppm Boron, T4 = 5 % Sucrose + 8 ppm Boron, T5 = 10 % Sucrose + 4 ppm Boron, T6 = 10 % Sucrose + 8 ppm Boron, T7 = 0.5 % Agar + 10 % Sucrose + 100 ppm Boron, T8 = 1 % Agar + 10 % Sucrose + 300 ppm Boron, T9 = 0.5 % Agar + 12 % Sucrose + 100 ppm Boron, T10 = 1 % Agar + 12 % Sucrose + 300 ppm Boron, T11 = Distilled water (Control)]. The viability of the pollen grains was evaluated following acetocarmine staining technique. The best germination of fresh pollen was recorded in treatment of 1 % Agar + 12 % Sucrose + 300 ppm Boron. A close perusal of the results of in vitro pollen germination showed that the lowest germination percentage was recorded in media with different concentration of sucrose and boron, not containing agar, and there is no germination of pollen takes place in distilled water. Significantly the highest in vitro pollen germination and pollen viability percentage was recorded in fresh pollen (95.00%) and as the duration of pollen storage increased in both the storage conditions, the in vitro pollen germination decreased. Of the two storage conditions of pollen, the higher in vitro germination percentage of pollen found in pollen stored in refrigerator compared to ambient storage condition. This study help in identification for culture media used to determine in vitro pollen germination and time period for pollen viability by in vitro pollen germination and pollen viability methods.