The rhabdomyosarcoma (RMS) is the most common type of soft tissue tumor in children and adolescents;yet only a few screens for oncogenic mutations have been conducted for RMS. To identify novel mutations andpotential therapeutic targets, we conducted a high-throughput Sequenom mass spectrometry-based analysis of238 known mutations in 19 oncogenes in 17 primary formalin-fixed paraffin-embedded RMS tissue samples andtwo RMS cell lines. Mutations were detected in 31.6% (6 of 19) of the RMS specimens. Specifically, mutations inthe NRAS gene were found in 27.3% (3 of 11) of embryonal RMS cases, while mutations in NRAS, HRAS, andPIK3CA genes were identified in 37.5% (3 of 8) of alveolar RMS (ARMS) cases; moreover, PIK3CA mutationswere found in 25% (2 of 8) of ARMS specimens. The results demonstrate that tumor profiling in archival tissuesamples is a useful tool for identifying diagnostic markers and potential therapeutic targets and suggests thatthese HRAS/ PIK3CA mutations play a critical role in the genesis of RMS.