The Drosophila melanogaster Z3314 line isolated from a Zvenigorodka (Ukraine) natural population is characterized by the manifestation and emergence of a wide spectrum of molecular aberrations. Among them, two types (the wing venation anomaly and violation of the leg segmentation) were the most represented. It was demonstrated that the frequency of manifestation (penetrance) and the expressiveness of these phenotypic aberrations increase with an increase in the temperature. When the Z3314 line is bred in the laboratory, autosomal visible rase (ra: 3–97.3) mutation, which leads to reduction of a part of dorso-central and scutellaria macrochaetae, was detected (isolated and identified). A number of genetic peculiarities that determined the consistency and prospects of the study were found during the mutation process study in the Z3314 line. The Z3314 line is characterized by a high frequency of the emergence of visible mutations in the X-Z3314 chromosome, which persisted for a long time of the breeding under laboratory conditions (from 2003 to 2011). Locus-specific high genetic instability in the singed locus in the X-Z3314 chromosome persisted from the moment of emergence of the first mutant alleles in 2006 until the end of the study. The emergence of mutations was observed both during the line breeding “inside” (in the case of brother–sister crossings) and after the crossings of the X-Z3314 chromosome carrier males with females of the С(1)DX,ywf/Y laboratory line with linked X chromosomes.