Background Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. Historically classified based on histology, advancements in molecular profiling have allowed for further sub-classification which has improved risk-stratification. While molecular profiling has improved the understanding of disease progression and risk, the molecular evolution of therapy resistance in patients with RMS has not been well described. Transcriptomic profiling of patients with high-risk, refractory RMS was undertaken with the goal of uncovering insights into the biology of treatment-resistant RMS. Procedure Formalin-fixed paraffin-embedded (FFPE) tissue samples from patients with refractory RMS who had samples archived at diagnosis and relapse were obtained. Histologic subtype and PAX3/7-FOXO1 fusion status was confirmed. Transcriptomic profiling of the pre- and post-treatment FFPE tissue samples was performed using the HTG Transcriptome Panel (HTG Molecular Diagnostics). Results We identified 11 patients with refractory RMS who had FFPE tissue samples archived at diagnosis and relapse following multimodality therapy. All patients were stratified as high-risk, including 5 with PAX3/7-FOXO1 fusion-positive RMS (FP-RMS) and 6 with PAX3/7-FOXO1 fusion-negative RMS (FN-RMS). The transcriptomic analysis revealed that the myogenesis pathway and markers associated with myogenic differentiation were enriched pre-treatment in patients with FP-RMS and enriched post-treatment in patients with FN-RMS. Conclusions Using a probe-based transcriptome panel to characterize matched pre- and post-treatment tissue samples from patients with RMS, we report that treatment-resistant RMS follows a fusion status-dependent evolutionary trajectory, marked by differential expression of myogenesis associated genes and myogenic differentiation markers.