China is the largest producer and exporter of royal jelly in the world. The high production of royal jelly in China is mainly attributed to a high royal jelly-producing lineage of honeybees (Apis mellifera) (HRJB). However, few studies have been conducted on the genetic characterization of HRJB. In this study, the mitochondrial DNA intergenic region between cytochrome oxidase I and II (COI-COII) and the mitochondrial NADH dehydrogenase subunit 2 sequences (ND2) were determined for 90 HRJB colonies, collected from the regions of China where HRJB originated, and 25 unimproved A. m. ligustica colonies from China. COI-COII sequence analysis revealed two mitotypes (C1 and C2d) in HRJB colonies and one mitotype (C1) in unimproved A. m. ligustica colonies. The main mitotype (C1) in HRJB accounted for 93% of the colonies. Based on ND2 sequences, four and two mitotypes were found in HRJB and unimproved A. m. ligustica colonies, respectively. Sequence alignment showed that nucleotides in three positions of the ND2 sequence were different between the main mitotype of HRJB and that of unimproved A. m. ligustica. Our study suggested that HRJB was bred from A. m. ligustica and possibly had genetic characteristics different from unimproved A. m. ligustica.