The LHX3 gene encodes LIM homeodomain class transcription factors that have important roles to play in pituitary and nervous system development. On the one hand, mutations of LHX3 are associated with deficiencies of growth hormone (GH), prolactin (PRL), luteotrophic hormone (LH), follicle-stimulating hormone (FSH) and thyroidstimulating hormone (TSH); on the other hand, mutations of LHX3 are also associated with combined pituitary hormone deficiency (CPHD) diseases in human and animal models. To date, few polymorphisms of the bovine LHX3 gene have been reported. In this study, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing methods were employed to screen the genetic variations within the bovine LHX3 gene in 802 Chinese indigenous cattle. The results revealed three novel single-nucleotide polymorphisms (SNPs): AY923832: g.7553G>A, 7631C>T and 7668C>G. Among them, a synonymous mutation of exon II was identified: GAG (Glu) >GAA (Glu) at position 72 aa (AY923832:g.7553G>A) of LHX3 (403aa) in the four Chinese bovine breeds. Significant statistical differences in genotypic frequencies for exon II and its flanking region of the LHX3 gene implied that the polymorphic locus was significantly associated with cattle breeds by the χ2-test (χ2 = 68.975, df = 6, P<0.001). Hence, the three novel SNPs not only extend the spectrum of genetic variations of the bovine LHX3 gene, but could also possibly contribute to conducting association analysis and evaluating these as genetic markers in bovine breeding and genetics, and CPHD detection.