We carried out genetic and cytogenetic analyses of X-ray-induced deleterious Z chromosomes that result in a soft and pliable (spli) phenotype in the silkworm, Bombyx mori . In a B. mori strain with a spli phenotype, we found the Z chromosome broken between the sch (1–21.5) and od (1–49.6) loci. We also found a chromosomal fragment bearing a fifth-chromosome locus for egg and eye pigmentation fused to a Z chromosome fragment. By means of fluorescence in situ hybridization using bacterial artificial chromosome clones as probes, we confirmed that the fused chromosome is composed of a fragment of chromosome 5 and a fragment of the Z chromosome. Moreover, a predicted gene, GA002017, the Bombyx ortholog of the Drosophila gene acj6 (Bmacj6), was completely deleted by the Z chromosome breakage event. The relationship between Bmacj6 and the spli phenotype is discussed.