The Gambusia holbrooki (eastern mosquitofish) reference genome will offer a crucial resource for understanding the evolution and adaptation of invasive freshwater fish species. The genome of G. holbrooki was assembled into two haplotypes through a phased assembly approach; however, only the primary haplotype was designated as the reference genome for annotation and downstream analyses. The entirety of the genome sequence was assembled into 24 contiguous chromosomal pseudomolecules and 1 mitochondrial genome. This chromosome-level assembly encompasses 0.67 Gb, composed of 421 contigs and 318 scaffolds, with contig and scaffold N50 values of 15.9 Mb and 29.6 Mb, respectively.