Purpose To investigate the correlation between DWI, intravoxel incoherent motion (IVIM), and hypoxia‐inducible factor 1‐alpha (HIF‐1α) expression in a nude mouse model of rhabdomyosarcoma based on imaging and pathological comparisons. Methods Human rhabdomyosarcoma‐derived (RD) cells were inoculated into the right thigh muscle of 20 BALB/c female nude mice. Mice were imaged using 3.0 Tesla MRI system. T 1 ‐weighted imaging, T 2 ‐weighted imaging, DWI, and IVIM images were obtained. ADW4.7 (GE Healthcare, ChicagoAQ34, IL, USA) was used for image processing of ADC, D slow , D fast , and f values. All parameter values were independently analyzed by 2 observers. Immunohistochemistry of HIF‐1α was performed. We used a specific image–pathology comparison method to ensure correct overlap between the image plane and the pathological section. Mann–Whitney U test or independent sample t test, Pearson or Spearman correlation test, the intragroup correlation coefficient, Kolmogorov–Smirnov test, and receiver operating characteristic curve were used. The correlation between DWI and intravoxel incoherent motion parameter values and HIF‐1α expression was determined. Results There were 10 mice in the low‐expression group and 7 in the high‐expression group. The ADC and D slow values were negatively correlated with HIF‐1α with correlation coefficients of −0.491 and − 0.702 ( P = 0.045 and 0.002). The f value positively correlated with HIF‐1α expression ( r = 0.485, P = 0.048). ADC, D slow , and f were significantly different between the high‐HIF‐1α expression tumors and the low‐HIF‐1α expression tumors. ADC showed the best predictive performance among all parameters (area under the curve = 0.652, sensitivity = 83.3%, specificity = 63.6%). Conclusion The parameter values of DWI and intravoxel incoherent motion can be used to evaluate the expression of HIF‐1α in rhabdomyosarcoma. ADC, D slow , and f value showed correlation with the expression of HIF‐1α.