Azolla is a floating fern with small leaves of the Azollaceae family. The classification of different Azolla species is highly controversial, making it difficult to identify Azolla species in Iran. Despite studies on the genetic diversity of Azolla , there is still no clear relationship between Azolla in Iran and its relation to other Azolla species. Azolla species present in Iran in central part of Anzali, Masal and Sari are unknown and various studies have identified it as A.filiculoides or A. pinnata . DNA barcoding is a molecular tool for rapid, reliable, and highly effective species identification that uses a specific DNA sequence in each species. In this study, sequencing of nuclear and chloroplastic regions (ITS, trnG-trnR, trnL-trnF) and SRAP and SCoT markers used to determine the genetic relationship between Azolla genotypes. Dendrograms based on barcode sequences in the gene bank and sequences of chloroplast and nuclear regions (ITS, trnG-trnR, trnL-trnF) showed that three samples, collected from Anzali wetland, Masal rice fields and Sari University of Agriculture and Natural Resources channel placed in a cluster with average similarity of 83% with A.caroliniana and zero genetic distance. The genetic distance of novel Azolla sp. with Iranian samples was zero but it is not included in this study as a diagnostic criterion because it was an unknown species. Also, Azolla samples of Iran with A. nilotica and A. pinnata had the highest genetic distance. The results of SCoT and SRAP markers data also showed that Azolla samples in Iran have high genetic similarity and are probably of the same species and have high genetic distance with A. pinnata . Therefore, according to the results of the present study and the results of previous researches, it can be concluded that the Azolla in Iran is not A.filiculoides and A.pinnata and is close to A.caroliniana . Keywords: Azolla , genetic diversity, DNA barcoding, SRAP, SCoT, A.caroliniana