In order to reveal the genetic variation and relationship of large old poplars, we collected 226 samples on 7 Populus species from 15 populations that distributed in southwest China, and molecular marker of the large old poplars was conducted by SSR technology, which will provide the basis for reasonable protection and utilization of large old poplars in this area. The results showed that: (1) 80 polymorphic loci were amplified by 7 pairs of primers. According to the values of the number of effective alleles, the genetic diversity among the large old poplars of 7 Populus species was in the order of P. schneideri > P. qamdoensis > P. kangdingensis > P. szechuanica var. tibetica > P. xiangchengensis > P. szechuanica > P. haoana, and the genetic diversity among populations within 5 Populus species were Daocheng population > Xiangcheng population > Yajiang population in P. xiangchengensis, Litang population > Xiangcheng population > Daocheng population in P. schneideri, Shangrila population > Deqen population in P. haoana, Markam population > Gonjo population > Qamdo population in P. qamdoensis, and Deqen population > Markam population in P. szechuanica var. tibetica, respectively. (2) The inbreeding coefficient (F_is) within population of P. xiangchengensis, P. schneideri, P. szechuanica var. tibetica, P. haoana and P. qamdoensis was -0.024 6, -0.253 5, 0.205 4, 0.240 1 and 0.029 2, respectively. The coefficient of genetic differentiation (Fst) was 0.156 1, 0.253 5, 0.128 8, 0.182 0 and 0.177 3, respectively, among the populations of P. xiangchengensis, P. schneideri, P. szechuanica var. tibetica, P. haoana and P. qamdoensis. Gene flow (N_m) among populations of P. xiangchengensis, P. szechuanica var. tibetica, P. haoana and P. qamdoensis was more than 1, except P. schneideri. (3) The genetic similarity coefficient among the 7 Populus species was ranged from 0.089 0 to 0.691 0, with the average of 0.402 0. The UPGMA clustering and Bayesian analysis both indicated that the 7 Populus species was divided into three groups. P. schneideri and P. kangdingensis comprised the first group, the second group consists of P. xiangchengensis and P. schneideri, while P. szechuanica var. tibetica, P. haoana and P. qamdoensis were at the third group. The UPGMA cluster result among populations revealed that the Litang population of P. schneideri was clustered with P. xiangchengensis, while the populations of other species were clustered closely. These results demonstrated that the large old poplars held high level of genetic variation, and the genetic variation mainly existed in different individuals within population, which means that the genetic diversity of large old poplars will decrease or loss results from the loss of individuals. Thus, it is essential to protect the large old poplar resources distributed in these areas.