Abstract: Due to the asynchronism during the process of megaspore mother cell meiosis, triploid poplars which were induced by chromosome doubling of megaspores through physical or chemical treatment could be either derived from the first-division restitution (FDR) or the second-division restitution (SDR). And it is difficult to identify the origin of the obtained triploids through their phenotypes. However, molecular marker analysis provides an efficient way to identify the origin of 2n female gametes. In this study, 87 triploid Populus tomentosa hybrids were induced by chromosome doubling of megaspores through high temperature treatment. Using these 87 triploids and their parents as material, five appropriate primers (female parent has heterozygous alleles which are different with alleles of male parent) with low recombination rate were selected to avoid the impact of homologous recombination on identifying the origin of 2n gametes. Based on these five SSR primers, results were shown that relative allele configurations at SSR loci of 2n female gametes in different triploids were inconsistent. Using the selected five SSR markers with low recombination rate, we identified the origin of 2n gametes of P. tomentosa triploid hybrids. It was shown that 35 triploids were originated from FDR 2n gametes, and the other 52 triploids were originated from SDR 2n gametes. Otherwise, five SSR primers with high recombination frequencies were detected and analyzed. The results confirmed that the randomly selected SSR primers used for the identification of origin were not completely reliable, only the SSR loci with low recombination frequencies can be used to identify the origin of 2n gametes accurately. The identification study of the origin of 2n gametes of triploid P. tomentosa is of great importance for the heterozygosity analysis of FDR and SDR 2n gametes, as well as the breeding strategy establishment for triploid P. tomentosa.