Gut microbiota of invasive Asian silver carp (SVCP) and indigenous planktivorous gizzard shad (GZSD) in Mississippi river basin were compared using 16S rRNA gene pyrosequencing. Analysis of more than 440 000 quality-filtered sequences obtained from the foregut and hindgut of GZSD and SVCP revealed high microbial diversity in these samples. GZSD hindgut (GZSD_H) samples (n=23) with >7000 operational taxonomy units (OTUs) exhibited the highest alpha-diversity indices followed by SVCP foregut (n=15), GZSD foregut (n=9) and SVCP hindgut (SVCP_H) (n=24). UniFrac distance-based non-metric multidimensional scaling (NMDS) analysis showed that the microbiota of GZSD_H and SVCP_H were clearly separated into two clusters: samples in the GZSD cluster were observed to vary by sampling location and samples in the SVCP cluster by sampling date. NMDS further revealed distinct microbial community between foregut to hindgut for individual GZSD and SVCP. Cyanobacteria, Proteobacteria, Actinobacteria and Bacteroidetes were detected as the predominant phyla regardless of fish or gut type. The high abundance of Cyanobacteria observed was possibly supported by their role as the fish’s major food source. Furthermore, unique and shared OTUs and OTUs in each gut type were identified, three OTUs from the order Bacteroidales, the genus Bacillariophyta and the genus Clostridium were found significantly more abundant in GZSD_H (14.9–22.8%) than in SVCP_H (0.13–4.1%) samples. These differences were presumably caused by the differences in the type of food sources including bacteria ingested, the gut morphology and digestion, and the physiological behavior between GZSD and SVCP.