[Objective] To deeply understand the molecular evolution pattern, pathogenicity, and drug resistance mechanism and provide a scientific basis for the prevention and control of Streptococcus equi subsp. zooepidemicus (SEZ), we compared the pathogenicity, drug resistance, and genome sequences of two SEZ strains (YLCD588 and HT222) isolated from donkeys in Xinjiang. [Methods] Whole-genome sequences of the two strains were determined by next-generation sequencing and a phylogenetic tree was constructed based on multilocus sequence typing (MLST) of sequencing data and existing sequences in the database. The virulence factor database (VFDB) (https://www.mgc.ac.cn/VFs/) and the center for genomic epidemiology (CGE) (http://genomicepidemiology.org) were used for annotation of the virulence and drug resistance genes of the two strains. The growth curves, antimicrobial susceptibility, and biofilm formation of the two strains were examined and compared. Mice were challenged with these strains separately and the pathogenicity of the strains was observed and evaluated. Then, histopathological changes and bacterial loads in the lung and spleen tissues of the morbid mice were observed and determined. [Results] Sequencing data showed that the genome sizes of YLCD588 and HT222 were 2 090 225 bp (1 905 coding sequences) and 2 105 005 bp (1 995 coding sequences), respectively. HT222 and YLCD588 carried 214 and 212 virulence genes, respectively. HT222 and YLCD588 had 235 and 233 drug resistance genes, respectively. YLCD588 was assigned to a novel sequence type (ST) 545 by MLST. The MLST phylogenetic tree indicated that YLCD588 was closely related to the goat-derived SEZ strain, while HT222 was closely related to the canine SEZ strain. YLCD588 displayed resistance to six antibiotics and HT222 exhibited resistance to four. The crystal violet assay and confocal laser scanning microscopy results showed that YLCD588 exhibited stronger biofilm formation than HT222 (P<0.05), whereas HT222 caused higher mortality rate (P<0.05), higher bacterial load (P<0.01), and severer pathological damage in mice than YLCD588. [Conclusion] The two SEZ strains exhibit distinct genomic characteristics, sequence types, pathogenicity, and drug resistance. HT222 possesses more drug resistance genes and virulence genes and exhibits stronger pathogenicity than YLCD588, while YLCD588 showcases stronger biofilm formation and drug resistance than HT222. These findings broaden the understanding about the molecular epidemiology, pathogenicity, and drug resistance of different SEZ strains from donkey and provide references for the effective control of the infection and spread of SEZ and clinical treatment of SEZ infection.