The spotted alfalfa aphid (SAA,Therioaphis trifolii) (Hemiptera: Aphididae) is a destructive pest of cultivated alfalfa (Medicago sativaL.) that leads to large financial losses in the livestock industry around the world. Here, we present a chromosome-scale genome assembly ofT. trifolii, the first genome assembly for the aphid subfamily Calaphidinae. Using PacBio long-read sequencing, Illumina sequencing, and Hi-C scaffolding techniques, a 541.26 Mb genome was generated, with 90.01% of the assembly anchored into eight scaffolds, and the contig and scaffold N50 are 2.54 Mb and 44.77 Mb, respectively. BUSCO assessment showed a completeness score of 96.6%. A total of 13,684 protein-coding genes were predicted. The high-quality genome assembly ofT. trifoliinot only provides a genomic resource for the more complete analysis of aphid evolution, but also provides insights into the ecological adaptation and insecticide resistance ofT. trifolii.