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As a member of MYLK family, MYLK4 gene may play a vital role in muscle development. In this study, one novel single-nucleotide polymorphism (SNP) was identified the bovine MYLK4 by sequencing pooled DNA samples (pool-Seq) and forced polymerase chain reaction-restriction fragment length polymorphism (forced PCR-RFLP) methods. Overall, we reported one mutation (SNP1) in the intron 10 region within the bovine MYLK4 gene in 559 individuals representing five main cattle breeds from China (Nanyang, NY; Qinchuan; Jiaxian, JX; Pinan cattle; and Caidamu cattle, CDM). Genotype AA and allele A were predominant in the QC, PN, and XN populations. Association analysis with growth traits in the QC breed showed that the animals with genotype GG had significantly greater chest breadth and hip width (P < 0.05). Meanwhile, the genotype GG was strongly associated with withers height and body length than those with genotype AA (P < 0.01 or P < 0.05) at 12 months in the NY breed. These statistical results exhibited that the MYLK4 gene might be a potential candidate gene to improve cattle's growth traits, and the SNP could be used as molecular markers in early marker-assisted selection (MAS) in beef cattle breeding program. [ABSTRACT FROM AUTHOR]