Abstract
High-quality reference genomes are critical resources for understanding biodiversity and supporting conservation efforts. We present the chromosome-level assembly of the Spanish moon moth, Actias isabellae (Graells, 1849), a nocturnal lepidopteran protected under the EU Habitats Directive. The assembly spans 0.56 Gb across 31 pseudomolecules, including the Z chromosome, with a contig N50 of 18.9 Mb and a scaffold N50 of 20.4 Mb. The mitochondrial genome was also assembled into a 15,247 bp circular sequence. Annotation identified 11,805 protein-coding genes and 2,238 non-coding genes, with a BUSCO completeness of over 94%. Notably, no W chromosome was detected, suggesting a ZZ/Z0 sex determination system. This reference genome provides an essential foundation for studying sex chromosome evolution in Lepidoptera and enables advanced population genomics monitoring of this protected species. More broadly, it contributes to ongoing efforts within the European Reference Genome Atlas and the Earth BioGenome Project to harness genomics for biodiversity conservation.