The tropical peatlands of southern Brazil are essential for the maintenance of the Atlantic Rain Forest, one of the 25 hotspots of biodiversity in the world. Although diazotrophic micro‐organisms are essential for the maintenance of this nitrogen limited ecosystem, so far studies have focused only on micro‐organisms involved in the carbon cycle. In this work, peat samples were collected from three tropical peatland regions during dry and rainy seasons and their chemical and microbial characteristics were evaluated. Our results showed that the structure of the diazotrophic communities in the Brazilian tropical peatlands differs in the evaluated seasons. The abundance of the genus Bradyrhizobium showed to be affected by rainfall and peat pH. Despite the shifts of the nitrogen‐fixing population in the tropical peatland caused by seasonality it showed to be constantly dominated by α‐Proteobacteria followed by Cyanobacteria. In addition, more than 50% of nifH gene sequences have not been classified, indicating the necessity for more studies in tropical peatland, since the reduction of N supply in the peatlands stimulates the recalcitrant organic matter decomposition performed by peatland micro‐organisms, influencing the C stock. Significance and Impact of the Study: This manuscript reports the first ecological study of tropical peatlands diazotrophic communities from the Brazilian Atlantic Rain Forest, one of the world's largest biodiversity hotspots. Based on culturing and culture‐independent approaches, our results showed alterations in the structure of the diazotrophic communities in different seasons. The genus Bradyrhizobium abundance was affected by rainfall and peat pH. In addition, the great number of unclassified nitrogen‐fixing bacteria indicated the need for additional studies to guide future mitigation actions, since the reduction of N supply in the peatlands stimulates the recalcitrant organic matter decomposition performed by peatland micro‐organisms, influencing the C stock.