BackgroundThe Western mosquitofish,Gambusia affinis, is a model for sex chromosome organization and evolution of female heterogamety. We previously identified aG. affinisfemale-specific marker, orthologous to the aminomethyl transferase (amt) gene of the related platyfish (Xiphophorus maculatus). Here, we have analyzed the structure and differentiation of theG. affinisW-chromosome, using a cytogenomics and bioinformatics approach.ResultsThe long arm of theG. affinisW-chromosome (Wq) is highly enriched in dispersed repetitive sequences, but neither heterochromatic nor epigenetically silenced by hypermethylation. In line with this, Wq sequences are highly transcribed, including an active nucleolus organizing region (NOR). Female-specific SNPs and evolutionary young transposable elements were highly enriched and dispersed along the W-chromosome long arm, suggesting constrained recombination. Wq copy number expanded elements also include female-specific transcribed sequences from theamtlocus with homology to TE. Collectively, theG. affinisW-chromosome is actively differentiating by sex-specific copy number expansion of transcribed TE-related elements, but not (yet) by extensive sequence divergence or gene decay.ConclusionsTheG. affinisW-chromosome exhibits characteristic genomic properties of an evolutionary young sex chromosome. Strikingly, the observed sex-specific changes in the genomic landscape are confined to the W long arm, which is separated from the rest of the W-chromosome by a neocentromere acquired during sex chromosome evolution and may thus have become functionally insulated. In contrast, W short arm sequences were apparently shielded from repeat-driven differentiation, retained Z-chromosome like genomic features, and may have preserved pseudo-autosomal properties.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12915-023-01607-0.