The diversity of eukaryotic and prokaryotic communities has been assessed by morphological and genetic approaches, which are used to characterize the microbiota in different environments. Here, planktonic prokaryotic and eukaryotic communities of the Araguaia River, located in the Central region of Brazil, were analyzed based on metabarcoding analysis of rRNA genes to evaluate the diversity of these groups in tropical floodplain lakes. Also, we tested their spatial concordance throughout the Araguaia river. Water samples were collected from 8 floodplain lakes in Araguaia River. The 16S and 18S rRNA genes were amplified and sequenced using Illumina MiSeq. For eukaryotes, 34,242 merged reads were obtained and 225 distinct OTUs were delineated, of which 106 OTUs were taxonomically classified. For prokaryotes, 26,426 sequences were obtained and 351 OTUs were detected. Of them, 231 were classified in at least one taxonomic category. The most representative eukaryotes belonged to Ciliophora, Chlorophyta and Charophyta. The prokaryotic phylum with the most OTUs classified were Proteobacteria, Actinobacteria and Bacteroidetes. The lakes did not show spatial concordance when comparing the similarity between their microbiota. The knowledge of freshwater biodiversity using DNA sequencing for important rivers, such as Araguaia River, can improve microbiota inventories of tropical biodiversity hotspots.