Diazotrophs are one of the most important microbial communities which play a key role in rhizosphere to enhance plant growth-promotion by supplying fixed nitrogen to the plants. The aim of present study was to analyze the frequency and diversity of diazotrophs in the rhizospheres of five genotypes of aromatic rice (Oryza sativa cultivars Nua Kalajeera, Ketekijoha, Nua Desheri, Nua Chandan and Kala Namak) grown in ten years old long-term aromatic rice-rice (LARR) cropping system under sub-humid tropical condition. In this study, it was observed that very less frequency of active diazotrophs (0-14.28%) in the rhizosphere of aromatic rice genotypes based on polymerase chain reaction (PCR)-based nifH gene amplification, dot blot hybridization and acetylene reduction assay (ARA). The following five isolates (B10, B12, B29, BK16, NKR16) from rhizosphere of aromatic rice and the reference strain Azospirillum brasilence showed nifH amplification and positive signal for dot blot. None of these isolates showed positive in ARA test except A. brasilence. Basic local alighnment search tool (BLAST) homology of nifH sequences of B10, B12, B29, BK16 and NKR16 matched with Gluconoacetobacter diazotrophicus, Pseudomonas stutzeri, Klebsiella sp., Klebsiella pneumoniae and Sinorhizobium meliloti, respectively. Whereas, isolates B10, B12 and BK16 were identified as Bacillus cereus, Enterobacter sp., and Lysinibacillus sp., respectively based on 16S-rDNA sequencing. As per nifH sequences, these diazotrophs (B10, B12, B29, BK16, NKR16) fall under alpha and gamma-proteobacter, whereas 16S-rDNA sequences revealed that they belonged to firmicutes and gamma-proteobacter. Multiple sequence alignment of nifH gene sequences showed wider variation among diazotrophs, however 3D structure of NifH protein revealed the less diversity among isolates. Moreover, restriction digestion of 16S-rDNA using tetracutters (Mspl, Mbol and HaeIII) deciphered a unique pattern among small population of diazotrophs. The results of this finding provided an interesting data and novel information of diazotroph diversity in LARR cropping system. Finally, the present study indicated that the continuous application of high dose of inorganic nitrogenous fertilizers (nitrogen at the rate of 60 & 80 kg N ha(-1) year(-1) wet and dry seasons, respectively) limit the frequency and diversity of rhizospheric diazotrophs in the LARR cropping system. (C) 2017 Elsevier B.V. All rights reserved.