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The diazotrophic communities play an important role in sustaining primary productivity through adding new nitrogen to oligotrophic marine ecosystems. Yet, their composition in the oligotrophic Indian Ocean is poorly understood. Here, we report the first observation of phylogenetic diversity and distribution of diazotrophs in the Eastern Indian Ocean (EIO) surface water (to 200 m) during the pre-southwest monsoon period. Through high throughput sequencing of nifH genes, we identified diverse groups of diazotrophs in the EIO including both non-cyanobacterial and cyanobacterial phylotypes. Proteobacteria (mainly Alpha-, Beta-, and Gamma-proteobacteria) were the most diverse and abundant groups within all the diazotrophs, which accounted for more than 86.9% of the total sequences. Cyanobacteria were also retrieved, and they were dominated by the filamentous non-heterocystous cyanobacteria Trichodesmium spp. Other cyanobacteria such as unicellular diazotrophic cyanobacteria were detected sporadically. Interestingly, our qPCR analysis demonstrated that the depth-integrated gene abundances of the diazotrophic communities exhibited spatial heterogeneity with Trichodesmium spp. appeared to be more abundant in the Bay of Bengal (p < 0.05), while Sagittula castanea (Alphaproteobacteria) was found to be more dominating in the equatorial region and offshores (p < 0.05). Non-metric multidimensional scaling analysis (NMDS) further confirmed distinct vertical and horizontal spatial variations in the EIO. Canonical correspondence analysis (CCA) indicated that temperature, salinity, and phosphate were the major environmental factors driving the distribution of the diazotroph communities. Overall, our study provides the first insight into the diversity and distribution of the diazotrophic communities in EIO. The findings from this study highlight distinct contributions of both non-cyanobacteria and cyanobacteria to N2 fixation. Moreover, our study reveals information that is critical for understanding spatial heterogeneity and distribution of diazotrophs, and their vital roles in nitrogen and carbon cycling. [ABSTRACT FROM AUTHOR]