<p>Complete ammonia oxidizers (comammox bacteria) can independently perform two-step nitrification and are crucial for nitrogen cycling. However, their niche differentiation across different types of aquatic environments remains poorly understood. Here, we investigated the niche differentiation of comammox bacteria in sediments from three typical water bodies (river, lake, and pond) in the middle reaches of the Yangtze River. The results showed that the <i>amoA</i> gene abundance of comammox Clade B was significantly greater in stagnant water bodies (pond: 2.89 × 10⁸ copies g^− 1) than in flowing water bodies (river: 5.58 × 10⁷ copies g^− 1, <i>P</i> &lt; 0.05; lake: 8.69 × 10⁷ copies g^− 1, <i>P</i> &lt; 0.01). Compared to non-rhizosphere sediments, rhizosphere sediments in the three water bodies showed higher mean <i>amoA</i> abundances of Clade A and Clade B. In addition, the mean total nitrification rate followed an order of the pond (1.744 ± 0.3045&#xa0;mg N kg^− 1d^− 1) &lt; the lake (2.033 ± 0.5871&#xa0;mg N kg^− 1d^− 1) &lt; the river (3.308 ± 0.7078&#xa0;mg N kg^− 1d^− 1), displaying an increasing trend with water fluidity, which was also reflected in comammox bacterial diversity. In the three water bodies, Clade A was identified as the predominant group within the comammox community, while Clade A.1 was the dominant functional clade in nitrification. Furthermore, the C/N ratio was identified as a key driver shaping niche differentiation among comammox clades in sediments. These results indicate that water flow shapes the niche differentiation of comammox bacteria in the middle reaches of the Yangtze River and provide new perspectives for aquatic ecological restoration.</p>