Objective To evaluate the sensitivity and specificity of regularly used immunohistochemical markers, including Vimentin (Vim), Desmin (Des), Myoglobin (MG), Myosin (MS), Smooth-muscle actin(SMA) and Sarcomeric actin ( Sr-A), in the diagnosis of rhabdomyosarcoma (RMS). Methods After resection, 24 RMSs and other childhood tumor specimens were fixed in 10% neutral-buffered formalin and embeded in paraffin. The immunohistochemical staining was performed by LSAB procedure. Heat-induced epitope retrieval of Des, MS, Sr-A was processed in order to enhence positive rate and positive strength. Results Vim, MG, MS,Des, Sr-A, SMA were arranged in the order of sensitivity from higher to lower. About specificity, Sr-A, Des, SMA,MG, Vim standed in a sequence from higher to lower (the data of MS is insufficient); Des, MG, Sr-A possessed higher experimental efficiency, followed by SMA, Vim in a succession. Conclusion Vim and MG are of the higher sensitivity but lower specificity. On the reverse, Sr-A and Des hold the better specificity but lower sensitivity.So the combination of multiple antibody reactions should be considered to improve the diagnostic ability in poorly differentiated RMS. According to the result of experimental efficiency, we suggest that the combination of Des, MG and Sr-A can make it possible to diagnose the majority of RMS clearly.