Japanese eel (Anguilla japonica) constitutes one of the most important food fish in Japan; accordingly, genome sequencing and linkage mapping have been conducted for the purpose of artificial cultivation. In the next stage, integration of genomic sequences within linkage groups (LG) is required to construct higher-resolution genetic markers for quantitative trait loci mapping and selective breeding of beneficial traits in farming. In order to identify LG1-linked scaffolds from the draft genome assembly (323,776 scaffolds) reported previously, we attempted to isolate chromosomes corresponding to LG1 by flow sorting and subsequent analyses. Initially, single chromosomes were randomly collected by chromosome sorting and subjected to whole-genome amplification (WGA). A total of 60 WGA samples were screened by PCR with primers for a known LG1-linked scaffold, and five positive WGA samples were sequenced by next-generation sequencing (NGS). Following reference mapping analysis of the NGS reads, four of the five WGA samples were found to be enriched by LG1-linked sequences. These samples were cytogenetically assigned to chromosome 5 by fluorescence in situ hybridization. Using blastn searches with 82,081 contigs constructed from the NGS reads of the four WGA samples as queries, 2323 scaffolds were identified as putative LG1-linked scaffolds from the draft genome assembly. The total length of the putative LG1-linked scaffolds was 99.0 Mb, comparable to the estimated DNA amounts of chromosome 5 (91.1 Mb). These results suggest that the methodology developed herein is applicable to isolate specific chromosome DNAs and integrate unanchored scaffold sequences onto a particular LG and chromosome even in teleost fishes, in which isolation of specific chromosomes by flow sorting is generally difficult owing to similar morphologies, sizes, and GC-contents among chromosomes in the genome. The putative LG1-linked scaffolds of Japanese eel contain a total of 6833 short tandem repeats which will be available for higher-resolution linkage mapping.