As one of the ecotypes of anadromous Coilia nasus, the freshwater resident Coilia nasus taihuensis is unable to conduct anadromous behavior due to the existence of physical barriers. C. nasus taihuensis can grow and reproduce independently in freshwater habitats such as Lake Taihu. Genetic differences in the genome have been demonstrated between anadromous C. nasus and non-anadromous C. nasus taihuensis due to geographical isolation and the significant difference in living environment. The genetic mechanisms related to environmental adaptation of C. nasus taihuensis were hindered by the lack of reference genomes. To conduct comprehensive and systematic analysis of the genomic characteristics of C. nasus taihuensis, a freshwater resident individual from Lake Taihu was selected as the experimental object. The first complete, gap-free reference genome of C. nasus taihuensis was constructed using a combination of PacBio (Pacific Biosciences) high-fidelity reads and ONT (Oxford Nanopore Technologies) ultra-long reads and Hi-C datasets. Based on a K-mer value of 21, we estimated the genome size as 612.76 Mb with a heterozygosity rate of 1.31%. The results of genome assembly showed a gap-free C. nasus taihuensis genome with an assembly size of 834.09 Mb. A contig N50 of 35.45 Mb was obtained through library construction, sequencing, assembly, chromosome mounting and gaps filling. Compared with the genome of cultured C. nasus, our gap-free genome substantially improved contiguity and completeness, where contig N50 increased from 1.6 Mb to 35.46 Mb. The integrity also increased from 87.1% to 91.9%, representing the highest quality genome. By Hi-C data, the assembled sequences were anchored and oriented onto all the 24 chromosomes with a total length of 829.28 Mb, covering 99.83% of the scaffold-level genome. After gap filling using ONT ultra-long reads, all 24 chromosomes were assembled without gaps, representing the highest assembly quality. BUSCO analysis based on the actinopterygii_odb10 database showed that 91.9% of the expected actinopterygii_odb10 genes (single-copy genes: 90%; duplicated genes: 1.9%) were identified as complete, suggesting that the assembled C. nasus taihuensis genome is highly complete. Furthermore, a total of 382.393 Mb of repetitive sequence, accounted for 45.85% of the genome. Using a combination of de novo prediction, protein homology and RNA-seq annotation, a total of 21730 protein-coding genes were identified. Approximately 99.71% (21666 genes) of the total predicted genes were assigned with at least functional annotation, showing a more complete annotation. Furthermore, the conservation synteny between C. nasus taihuensis and C. nasus was compared to validate the chromosome assembly, and the results showed that the genomic sequences of C. nasus and C. nasus taihuensis were highly consistent (96.95%). Such highly conserved synteny and strict correspondence of chromosome assignment indicated a close genetic relationship between the two ecotypes. The high-quality gap-free assembled genome of C. nasus taihuensis can provide material for studying the freshwater adaptability of C. nasus and accumulate basic data for further studies on the population genetics of C. nasus.