The wide occurrence of potato genotypes characterized by male sterility considerably complicates the realization of breeding programs. Therefore, male fertility in potato germplasm requires assessment as an important task. In the fresh study, 57 breeding lines from the Russian Potato Research Center and 85 cultivars of different origins underwent acetocarmine staining and in vitro germination assessment. With acetocarmine staining, 22 samples exhibited with tetrad sterility. In the remaining 120 samples, stained pollens vary from 0% to 88.7%, and germination varies from 0% to 19.4%. The pollen fertility assessment data obtained through acetocarmine staining and in vitro germination differed significantly. In the studied potato accessions, acetocarmine staining provided 18.3% sterile samples, while 50.7% by in vitro germination. The highest stained pollens (>80%) appeared in five potato accessions, i.e., 3004-7 (88.7%), 3000-32 (86.5%), 21.32-1 (81.8%), 17.32-1 (80.3%), and cultivar Garant (86.8%). In most potato accessions, the colored pollen grains ranged from 40% to 70%. The maximum percent of pollen germination occurred in the sample, 21.32-1 (19.4%). However, seven accessions (21.32-1, 18.5-17, 20.3-6, 20.33-8, and cultivars Edison, Queen Anne, and Garant) showed more than 10% pollen germination. During the growing season of 2022, the weather was hotter and drier compared with past long-term data, which could affect a decrease in the ability of pollens to germinate. The obtained results on male fertility will help in the study of the genetic control of pollen fertility and a further improvement in the breeding of Solanum tuberosum L.