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The Japanese Eel Anguilla japonica is an important migratory fish with great commercial and conservational significance in Asia. The Pearl River in southern China provides important natural growing and rearing grounds for glass eel recruits, but there is very limited information on their population characteristics. We studied the resource status and habitat preferences of Japanese Eels in the Pearl River from 2015 to 2018. In total, 181 Japanese Eels (89 males and 92 females) were collected during the study period. In terms of frequency of occurrence, Japanese Eels contributed less than 1% of the total number of collected fish specimens. Most of the eels were between 255 and 600 mm TL. Redundancy analysis indicated that eels were mainly distributed in the downstream portion of the river and preferred more complex habitats characterized by larger river fractals, higher coefficients of fluvial facies, and greater river widths. Adults preferred to inhabit areas with larger fractals and river widths, whereas juveniles inhabited areas with higher coefficients of fluvial facies. Young‐of‐the‐year eels preferred habitats near the estuary and with high water salinity. Dams have blocked the eels' migration routes and affected their spatial distribution, in turn leading to a reduction in the population. This study was part of a long‐term investigation of wild Japanese Eels in the Pearl River, and the results showed that the Japanese Eel resource in southern China is extremely scarce and its conservation is greatly needed. [ABSTRACT FROM AUTHOR]