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Simple Summary: Urbanization has been regarded as a major threat to fish diversity in urban waters. Xinglinwan Reservoir, a lentic brackish ecosystem with a distinct salinity gradient in southeastern China, is experiencing rapid urbanization, and there are no reports on fish diversity here. Environmental DNA metabarcoding (eDNA) has been applied to biodiversity assessments in aquatic ecosystems, but there has been limited work on fish diversity in lentic brackish ecosystems. We assessed the fish diversity and spatial distribution along a salinity gradient in Xinglinwan Reservoir, combining eDNA and the traditional survey method (TSM). This study provides realistic pictures of fish species assemblages in Xinglinwan Reservoir and tests the efficiency of eDNA for assessing fish diversity in lentic brackish ecosystems. Our results contribute to the conservation of fish resources in Xinglinwan Reservoir and advance the application of eDNA in lentic brackish ecosystems. With the decrease in fish diversity, the monitoring of fish has become a current research hotspot. Here, the fish diversity and spatial distribution along a salinity gradient (0.28‰–3.32‰) in Xinglinwan Reservoir, China, were investigated using eDNA and the TSM. With the combination of both methods, 12 species from 6 orders, 9 families, and 11 genera were observed. The invasive species Oreochromis niloticus had the highest relative abundance, accounting for 56.91% in the TSM and 89.80% in eDNA, respectively. eDNA detected 83.33% of the species detected using the TSM, and an additional six species (50.0% of the total species) were detected via only eDNA. Moreover, eDNA supplemented the unavailable abundance data from the TSM. The TSM revealed that the diversity and relative abundance of fish inhabiting seawater and brackish water increased with an increase in salinity, but this was not found using eDNA. Thus, our study highlights that Xinglinwan Reservoir has a low species richness and is suffering from the great harm of ecological invasion. Our findings also show that eDNA could be reliable for providing a comprehensive picture of fish, but discriminating the spatial heterogeneity of fish communities is a challenge for its widespread adoption in lentic brackish systems. [ABSTRACT FROM AUTHOR]