Abstract: The effect of boron on the migration and colonization of rhizobia in Gannong No.5 alfalfa (Medicago sativa Gannong No.5) tissues and subsequently seedling growth were investigated by root drenching using two cyan fluorescent protein (CFP) tagged rhizobia; Ensifer meliloti 12531f (12531f) and Ensifer meliloti GN5f (gn5f) containing different boron concentrations. The results showed that the best supplementation levels were less than 1 and 100 mg/L boron for 12531f and gn5f respectively. The optimum boron level enhanced the colonization of both rhizobia in alfalfa roots; colonization densities of 2184.99 and 58307.11 cfu/g were achieved by using 100 mg/L boron co-inoculated 12531f and 0.5 mg/L boron co-inoculated gn5f, respectively. Rhizobia were able to migrate to the aerial tissues and colonize lower stems and leaves through application of 1 mg/L boron co-inoculated 12531f and 100 mg/L boron co-inoculated gn5f, respectively. No fluorescent tagged rhizobia were detected in the control treatment. Individual plant root nodule number, nodule weight, leaf number, plant height, root length, aerial fresh weight, aerial dry weight, root fresh weight and root dry weight were increased by 21.31%-909.28% for the 1 mg/L boron co-inoculated 12531f treatment compared with control and zero boron innoculation. These traits were increased by 15.07%-1424.24% for the 100 mg/L boron co-inoculated gn5f treatment compared with control and zero boron inoculation treatments. Leaf chlorophyll content was reduced by 12531f without boron whereas 1 mg/L boron co-inoculated 12531f significantly increased the leaf chlorophyll content by 18.31% and 36.86% compared with the control and zero boron inoculation, respectively (P<0.05). Boron supplemented gn5f enhanced the leaf chlorophyll content; 100 mg/L boron co-inoculated gn5f increased leaf chlorophyll content by 24.41% and 12.92% compared with control and zero born inoculation, respectively (P<0.05). The results suggest that 1 mg/L boron co-inoculation with 12531f, 100 mg/L boron co-inoculation with gn5f promoted the migration and colonization of rhizobia in alfalfa seedlings, enhancing growth, indicating possible benefits for alfalfa cultivation.