Ferric-chelate reductase which functions in the reduction of ferric to ferrous iron on root surface is a critical protein for iron homeostasis in strategy I plants.LeFRO1 is a major ferric-chelate reductase involved in iron uptake in tomato.To identify the natural variations of LeFRO1 and to assess their effect on the ferric-chelate reductase activity,we cloned the coding sequences of LeFRO1 from 16 tomato varieties collected from different regions,and detected three types of LeFRO1(LeFR01~(MM),LeFRO1~(Ailsa) and LeFRO1~(Monita)) with five amino acid variations at the positions 21,24,112,195 and 582.Enzyme activity assay revealed that the three types of LeFRO1 possessed different ferric-chelate reductase activity(LeFRO1~(Ailsa)>LeFRO1~(MM)>LeFRO1~(Monita)).The 112th amino acid residue Ala of LeFRO1 is critical for maintaining the high activity of ferric-chelate reductase,because modification of this amino acid resulted in a significant reduction of enzyme activity.Further,we showed that the combination of the amino acid residue Ile at the site 24 with Lys at the site 582 played a positive role in the enzyme activity of LeFRO1.In conclusion,the findings are helpful to understand the natural adaptation mechanisms of plants to iron-limiting stress,and may provide new knowledge to select and manipulate LeFRO1 for improving the iron deficiency tolerance in tomato. Ferric-chelate reductase which functions in the reduction of ferric to ferrous iron on root surface is a critical protein for iron homeostasis in strategy I plants. LEFRO1 is a major ferric-chelate reductase involved in iron uptake in tomato. To identify the natural variations of LeFRO1 and to assess their effect on the ferric-chelate reductase activity, we cloned the coding sequences of LeFRO1 from 16 tomato strains collected from different regions, and detected three types of LeFRO1 (LeFR01 ~ (MM), LeFRO1 ~ (Ailsa) and LeFRO1 ~ Monita) with five amino acid variations at the positions 21, 24, 112, 195 and 582. Enzyme activity assay revealed that the three types of LeFRO1 possessed different ferric-chelate reductase activity (LeFRO1 ~ (Ailsa)> LeFRO1 ~ (MM)> LeFRO1 ~ (Monita)). The 112th amino acid residue Ala of LeFRO1 is critical for maintaining the high activity of ferric-chelate reductase, because modification of this amino acid resulted in a significant reduction of enzyme activity .Further, we showed tha t the combination of the amino acid residue Ile at the site 24 with Lys at the site 582 played a positive role in the enzyme activity of LeFROl. In conclusion, the findings are helpful to understand the natural adaptation mechanisms of plants to iron-limiting stress , and may provide new knowledge to select and manipulate LeFRO1 for improving the iron deficiency tolerance in tomato.