This study was conducted to contrast two staining techniques for determining pollen viability in sugarcane. At three diverse times (6:30, 8:00, and 9:30 a.m.), pollen from seven sugarcane genotypes was gathered, and its vitality was assessed using Lactophenol blue and Iodine stains. For every genotype, using a drop of the respective stain, three others were crushed on a glass slide. Pollen viability percentages were determined using an optical microscope. Variance analysis was used to analyze the factors (staining methods, genotypes, and times) and their interaction, and the means were compared. Lactophenol blue staining proved more sensitive than Iodine staining in detecting the natural decline in pollen viability in sugarcane. This study underscores the effectiveness of Lactophenol as a reliable medium for assessing pollen viability, offering a more accurate representation of the physiological state of pollen grains at various times (6:30, 8:00, and 9:30 a.m.) for different sugarcane genotypes. The genotypes exhibited variations in pollen grain viability percentage during time intervals. Genotypes G2004/27 and EH78-26-11 showed superior pollen grain vitality in both staining methods at early time, whereas genotype G2003/47recorded the lowest values at the later time.