Phytoseiulus persimilis and Neoseiulus californicus (Phytoseiidae, Arachnida) are important predatory mites in agriculture, mainly preying on harmful mites like Tetranychus urticae for biological control. P. persimilis, native to the Mediterranean coast, is a commercially available natural enemy with a specialized diet and high control efficiency. N. californicus, widely distributed in China, has strong insecticide resistance and starvation tolerance, and can also control small insects such as thrips. This study used de novo whole-genome sequencing (combining long-read and short-read data) to assemble the genomes of the two mites, evaluated by Scaffold N50 and BUSCO. High-quality genome maps were obtained: P. persimilis and N. californicus have genome sizes of 190 Mb and 182 Mb, respectively, with Scaffold N50 >45 Mb and chromosome-level genome assembly and annotation completeness >90%. Synteny analysis showed high conservation in gene arrangement but some chromosome rearrangements. Gene family analysis identified expanded families related to insecticide resistance and stress tolerance in N. californicus. Both mites experienced enhanced negative selection. In summary, this study comprehensively employed multi-dimensional analytical approaches, including comparative genomics, evolutionary genomics, and functional gene annotation, to systematically dissect the genomic structural characteristics, gene family evolution patterns, and species-specific genetic variations o P. persimilis and N. californicus. It further elucidated the genomic divergence between these two important predatory mites shaped during long-term evolution and its potential implications for ecological adaptation. The findings not only provide a novel perspective for uncovering the molecular regulatory mechanisms underlying key biological processes such as development, reproduction, and stress resistance in these two predatory mites, but also lay a solid molecular foundation and important theoretical support for subsequent applied research including their genetic improvement, optimization of large-scale efficient rearing techniques, and enhancement of field biocontrol efficacy.