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Background: In a genetic breeding program, understanding the viability of pollen germination and pollen tube growth is an essential factor for artificial hybridization. Currently, little is known about the influence of sucrose, boric acid concentrations and calcium chloride on pollen viability in alfalfa. This study is valuable for providing information about pollen germination requirements to support further improvement of artificial pollination and breeding programs in alfalfa. Methods: The single-factor experimental and an orthogonal design method (L16 [4]3) was used to compare the effectiveness of key factors (e.g., concentrations of sucrose, boric acid and calcium chloride) to determine the pollen vigor of Medicago varia Martin. ‘Caoyuan No.1’and M. ruthenica L. Trautv. ‘zhilixing’ and to optimize the culture medium for in vitro pollen germination. Four different varieties were chosen to compare the diurnal variation of pollen vigor. Result: The four factors that influenced pollen germination in Medicago varia Martin. ‘Caoyuan No.1’and M. ruthenica L. Trautv. ‘zhilixing’ in order of significance are as follows: sucrose greater than H3BO3 greater than CaCl2. These variables influenced pollen tube growth in the following order: sucrose greater than H3BO3 greater than CaCl2. The optimal culture medium for promoting pollen vigor of Medicago varia Martin.‘Caoyuan No.1’ was 10% sucrose, 300 mon/L H3BO3 and 100 mon/L CaCl2; that of M. ruthenica L. Trautv. ‘zhilixing’ was 10% sucrose, 200 mon/L H3BO3 and 50 mon/L CaCl2. Among several species of alfalfa, ‘Caoyuan No.1’ shows the highest pollen vigor and the daily pollen activity curves of all varieties showed a single peak type.