The AMP-activated protein kinase (AMPK) is an evolutionarily conserved sensor of cellular and systemic energy balance. PRKAB1, the gene that encodes the beta1 regulatory subunit of AMPK, has been shown to be highly involved in the glycogen metabolism. To date, several mutations affecting function of human PRKAB1 have been identified but few studies have shown a complete description of the variability of bovine PRKAB1. In the present study, we report the investigation of PRKAB1 genetic polymorphism in a sample of 811 Chinese indigenous bovine individuals. The screening of the coding regions with their intron-exon boundaries and the proximal flanking regions was performed using a PCR-SSCP strategy. Following sequence analysis revealed 29 single nucleotide polymorphisms (SNPs) of bovine PRKAB1 gene. Four were in the 5'-UTR; eight were in the coding region, among which one was nonsense mutant,one was missense mutant, six were synonymous mutation; and seventeen were in the introns. We also investigated haplotype frequencies and linkage disequilibrium (LD) coefficients for these SNPs in three Chinese indigenous cattle breeds. Three LD blocks were found in Nanyang cattle and four common haplotypes were identified based on five SNPs, with the most common haplotypes (GGTCC) occurring at a frequency of 82.7%. In Qinchuan cattle, one major block and two of the four possible haplotypes were found, one main haplotypes (CG) accounted for 93.8% of all possible marker combinations. In Jiaxian cattle, two LD blocks were found and four common haplotypes were identified based on four SNPs, the most common haplotypes (CCGT) accounted for 77.4% of the total. Variations detected here might have an impact on PRKAB1 activity and function and underpin the development of gene markers for bovine energy balance and in the glycogen metabolism.