Polyploid breeding is an efficient method for breeding new varieties in rubber trees (Hevea brasiliensis Müll. Arg.). In order to reveal the genetic origin of 14 tetraploid lines, 25 simple sequence repeat markers were subjected for analyzing the diversity within this study. The leaf anatomy structure of diploid, triploid and tetraploid lines were analyzed using two section methods to evaluate the difference between ploidy. As a result, all 14 tetraploids are derived from the same clone or full siblings, and the diploid line Reyan73397 serves as one of the parental lines. The thickness of the leaves, upper epidermis, palisade tissue and sponge tissue between the diploid, triploid and tetraploid of the rubber tree was significantly different and increased with the increasing of the ploidy. The thickness of diploid, triploid and tetraploid fresh leaves are 122.63~195.68μm, 163.49~232.96μm, 169.16~239.30μm, and the thickness of the paraffin leaves are 110.04~161.18μm, 140.34~198.59μm and 136.78~240.95μm respectively. The demarcation of fresh leaf thickness of 177.81 μm or the paraffin wax thickness of 160.70 μm can be used to identify tetraploid from mixed population of diploid and tetraploid. The rank of Cell Tense Ratio and Palisade tissue /Spongy tissue ratio in different ploidy was as follows:triploid＞tetraploid ＞diploid, the rank of Spongy Ratio in different ploidy was as follows: diploid＞tetraploid＞ triploid. Takentogether, the findings derived from this study are valuable for clarifying the polyploidy resources and future breeding in polyploidy rubber trees.