[Objective] Since hyaluronic acid (HA) biosynthesis pathway and the related genes in Streptococcus equi subsp. zooepidemicus have been thoroughly studied, it is of great significance of exploring a kind of strategy to identify new genes related to the synthesis of HA. [Methods] strains phenotype defect were screened using the randomly incorporation of the suicide vector pSET4s::sacB in host genomes and a mutant library was constructed. Integration loci were detected through ligation mediated PCR (LM-PCR) and verified by gene deletion and complementation, as well as complete genomic sequencing. [Results] A mutant library containing 150 mutants strains phenotype defect was constructed. We detected that the temperature sensitive vector was incorporating at 458 960 loci in Streptococcus zooepidemicus genomes and disrupting gene lacC which encodes 6-phosphate kinase. ΔlacC was obtained through markerless gene deletion system, and the phenotype analysis showed that ΔlacC still displayed the mucoid phenotype that was not the same as M1. The further complete genomic sequencing showed that base G in the locus of 206 613 in M1 mutant genome was lost, which was located in hasA gene encoding hyaluronate synthase and leading to frameshift mutation of hasA. What’s more, when complementation of hasA in M1 mutant, interestingly, M1 mutant rescued the capsule synthesis ability and displayed mucoid phenotype. [Conclusion] The result indicated that the loss of capsule synthesis ability of M1 mutant was the result of the loss of hasA gene function, which had nothing to do with the lack of lacC gene function. This study preliminarily established the strategies to high throughput screening new genes related to synthesis of HA in Streptococcus zooepidemicus, which laid a foundation for mining new genes in future.