Crossbreeding is a multi-stage process with inherent challenges and risks in developing new varieties. Success hinges on selecting highly fertile parents. In species like lisianthus, uncertainty persists regarding the optimal methods for assessing pollen quality, which is crucial for evaluating pollen parent fertility. This study seeks to identify the most reliable techniques for this purpose. Fresh and dead pollen from four lisianthus (Eustoma grandiflorum) varieties was used. The dead pollen was obtained by thermal inactivation. Five chemical staining methods (iodine-potassium iodide, 2,3,5-triphenyltetrazolium chloride – TTC, lactophenol cotton blue, safranin, acetocarmine) were employed to assess pollen viability, and two biological methods (Petri dishes, hanging drops) were used to determine the germination rate. Four solid medium cultures were employed in Petri dishes, while the hanging drop utilised four liquid medium cultures. Thirteen tests were conducted for each variety, evaluating fresh and dead pollen. The study found significant variations in pollen quality among lisianthus varieties and methods. Fresh pollen showed viability rates ranging from 56.87% to 99.41% and germination rates from 0.20% to 45.11%. TTC exhibited the lowest viability rate across all varieties, while the highest germination rate was observed in the liquid culture medium with only boric acid and PEG1500. Notably, TTC was the sole viability method that did not stain dead pollen, and no germination occurred in any method for dead pollen. TTC is the most reliable staining method, and a liquid culture medium with boric acid and PEG1500 effectively determines lisianthus pollen quality. Varying boric acid and PEG1500 concentrations are advisable.