To investigate the evolutionary dynamics and characteristic of the molecular transmission networks of HIV-1 CRF01_AE in Nanjing.Viral samples were collected from 580 newly diagnosed HIV-1-infected patients. HIV-1 pol sequences were obtained and used for for molecular evolutionary analyses. The ML trees were constructed by MEGA 6.0 using under GTR+ G + I model with 1000 bootstrap replicates. The emergence and estimation of tMRCA and the evolutionary rate of the different CRF01_AE clusters were inferred using Bayesian phylogenetic analysis approaches implemented in the BEAST package. Pairwise genetic distances were calculated under the Tamura-Nei 93 model, a genetic distance threshold of ≤1.2% was used to identify potential transmission clusters. Network diagrams were plotted using Cytoscape 3.3.0.Of these HIV-1-infected patients, 551 (91.5%) were males. The largest number of infections were attributed to homosexual (462, 79.7%). A total of 518 full-length pol genes were successfully amplified, based on the phylogenetic analysis CRF01_AE was the most predominantly circulating strain (45.0%, 233/518). As shown in the ML tree, three distinct clusters were observed. The 'Nanjing lineage' 1, 2, 3 has an estimated tMRCA around1996.61, 1993.61, 1984.61 respectively. Of 233 Nanjing sequences, 123 (55.2%) distributed in 30 molecular clusters, average Links/node was 7.8 with range (1-33), most of Nanjing strains shared links with local strains.HIV-1 CRF01_AE was the most predominantly circulating strain, the epidemic of CRF01_AE in Nanjing was driven by multiple clusters of HIV-1 strains, and most CRF01_AE stains in our study were estimated to have originated in China in the 1990s.