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The quality and safety of milk is challenged by cow mastitis, and the value of somatic cell score (SCS) in milk is closely related to the occurrence of mastitis. This study aimed to analyze the genetic characteristics of SCS across the first three parities in Chinese Holstein cattle, as well as to investigate potential candidate genes and biological processes that may play a potential role in the progress of cow mastitis. In this respect, we evaluated genetic parameters and conducted a genome-wide association study based on the test-day records of SCS for Chinese Holstein cows; we also validated key candidate genes using a quantitative reverse transcription PCR (RT-qPCR) experiment in primary bovine mammary epithelial cells (bMECs). The heritability of the SCS 305-day performance in milk varied between 0.07 and 0.24, and decreased with increasing parity. As the time interval grew larger, the genetic and permanent environmental correlations with the number of days in milk (DIM) weakened. Six significant single-nucleotide polymorphisms (SNPs) were identified in the association analysis, one of which was located within the exonic region of <i>CD44</i>. This exon-associated SNP may modify the activity of the protein encoded by the <i>CD44</i>. A total of 32 genes within the two hundred kilobase (kb) range of significant SNPs were detected, and these genes were markedly enriched in eight Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and 22 biological processes, mainly participating in the progress of transmembrane transport, inflammatory factor regulation, cellular responses, the Toll-like receptor signaling pathway, and the MAPK signaling pathway. Nine genes, including the <i>PKD2</i>, <i>KCNAB1</i>, <i>SLC35A4</i>, <i>SPP1</i>, <i>IBSP</i>, <i>CD14</i>, <i>CD44</i>, <i>MAPK10</i>, and <i>ABCG2</i> genes, were selected as candidate genes that could have critical functions in cow mastitis. These findings can serve as a foundation for molecular breeding and as valuable data for reducing the incidence of mastitis of Chinese Holstein cattle at the molecular level.