Hormone-sensitive lipase (HSL) was considered as an essential enzyme in glucolipid metabolism. It has been proposed to be a lead candidate gene for genetic markers of lipid deposition in livestock. The aim of this study was to identify sequence variants (SVs) of the bovine HSL gene and evaluate the relations to intramuscular fat in two indigenous Chinese beef cattle breeds. Expression analysis by quantitative real-time polymerase chain reactions (qPCR) indicated that expression levels of bovine HSL gene were highest in the perirenal fat and heart within two different age stage (adult and calf), respectively. Five SVs were identified by direct DNA sequencing, which included four missense mutations (g.16563C>T, g.16734G>A, g.16896A>G, g.17388G>T) in exon 8 and a synonymous mutation (g.17402C>T) in exon 9. Population genetic analysis showed that except for g.16563C>T and g.17402C>T, all the other detected SVs strongly affected the bovine intramuscular fat content (P <0.01 or P <0.05). The individuals with Hap5/5 diplotypes (CC-GG-GG-GG-CC) was highly significantly associated with intramuscular fat content than the other diplotypes (P <0.01). The above results suggested that the HSL gene can used as potential candidate markers gene for the beef breed improvement through marker assisted selection in Chinese cattle breeds.