Abstract
<title>Abstract</title> <p> The Eastern mosquitofish, <italic>Gambusia holbrooki</italic> , is a small livebearing fish native to the southeastern United States that has been introduced globally and is now considered one of the world's most invasive freshwater species. In Australia, established populations have undergone decades of adaptation to diverse thermal and ecological environments. Here we present a fully phased chromosome-level genome assembly of <italic>G. holbrooki</italic> from Townsville, Queensland, Australia, generated using PacBio HiFi long-read and Hi-C chromatin conformation sequencing. The assembly comprises 24 chromosomes and two haplotypes of approximately 676 Mb each, with contig and scaffold N50 values exceeding 26 Mb and 29 Mb, respectively. BUSCO completeness exceeded 98.4% for both haplotypes and Merqury quality values exceeded Q66. Telomeric repeat motifs were identified at both ends of 13 (HAP1) and 10 (HAP2) chromosome-scale scaffolds, with a further 9 (HAP1) and 7 (HAP2) chromosomes carrying signal at one end, indicating near-complete end-to-end assembly for most chromosomes. This resource provides a high-quality genomic reference for studying the invasion biology, local adaptation, sexual selection and evolutionary genomics of invasive fishes, and a foundation for future comparative analyses across globally distributed populations. </p>