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High-throughput sequencing technologies play important roles in the study of animal enteric microorganisms. Fecal samples collected from 3 strains (9 samples) of specific-pathogen-free (SPF) ducks raised in an isolator. Duck intestinal bacterial flora were analyzed by sequencing distinct regions of the 16S rRNA genes using an Illumina HiSeq2500 platform. The result showed the most abundant primary enteric microbial phyla of 3 different strains of 70-week-old SPF ducks were Firmicutes, Proteobacteria, and Bacteroidetes, the most abundant primary enteric microbial order were Clostridiales, Lactobacillales, and Aeromonadales, and the most abundant primary enteric microbial genera were Bacteroides, Comamonas, and Enterococcus. In addition, the 3 duck strains harbored different compositions of the microorganisms, but these differences were not significant. Duck intestinal bacterial flora were analyzed using a high-throughput sequencing approach to further understand the distribution of intestinal flora and the biology of SPF ducks to ultimately benefit the purification of SPF ducks.