Organisms are remarkably adapted to diverse environments by specialized metabolisms, morphology, or behaviors. To address the molecular mechanisms underlying environmental adaptation, a Drosophila melanogaster line, termed “Dark-fly” has been maintained in constant dark conditions for 57 years (1400 generations). Dark-fly exhibited higher fecundity in dark than in light conditions, indicating that Dark-fly possesses some traits advantageous in darkness. Using next-generation sequencing technology, the whole genome sequence of Dark-fly was obtained and approximately 220,000 single nucleotide polymorphisms (SNPs) and 4,700 insertions or deletions (InDels) were unique for the Dark-fly genome compared to the genome of the Oregon-R-S strain, a control strain. 1.8% of SNPs were classified as non-synonymous SNPs (nsSNPs: i.e., they alter the amino acid sequence of gene products). Among them, we detected 28 nonsense mutations (i.e., they produce a stop codon in the protein sequence) in the Dark-fly genome.