Objective To improve the gene targeting efficiency with C57BL/6 embryonic stem (ES) cells. Methods Three different genetically modified C57BL/6 ES cell lines, named TLX3, Ai3K and SL, were microinjected into ICR, B6(Cg)-Tyr^c-2J and BALB/c mouse blastocysts, respectively. The efficiency was statistically evaluated according to three aspects: blastocyst collection, chimera production and germline transmission. Results None of the three ES cell lines was germline transmitted with B6(Cg)-Tyr^c-2J mice as blastocyst donors, while it was achieved with both BALB/c and ICR mouse blastocysts. Compared in the aspect of blastocysts collection, ICR mouse was much better than BALB/c mouse (P<0.05), and the chimera production efficiency of ICR mouse was comparable to that of BALB/c mouse (P=0.115). As to the germline transmission efficiency, that of BALB/c mice is significantly higher than that of the ICR mice (P< 0.01). Conclusions The germline transmission efficiency of BALB/c mouse is highest among these three mouse strains. However, it has the disadvantages of blastocyst collection, developmental delay and zona pellucida fragility, compared with ICR mouse. Therefore, ICR mouse is also a good candidate as blastocyst donor for embryonic stem cell microinjection.