Post stroke muscle stiffness is a common problem, which left untreated can lead to disabling muscle contractures. The purpose of this pilot study was to evaluate the feasibility of bi-exponential T1ρ mapping in patients with arm muscle stiffness after stroke and its ability to measure treatment related changes in muscle glycosaminoglycans (GAGs). Five patients with muscle stiffness after stroke and 5 healthy controls were recruited for imaging of the upper arm with 3D-T1ρ mapping. Patients were scanned before and after treatment with hyaluronidase injections, whereas the controls were scanned once. Wilcoxon Mann-Whitney tests compared patients vs. controls and patients pre-treatment vs. post-treatment. With bi-exponential modeling, the long component, T1ρl was significantly longer in the patients (biceps P = 0.01; triceps P = 0.004) compared to controls. There was also a significant difference in the signal fractions of the long and short components (biceps P = 0.03, triceps P = 0.04). The results suggest that muscle stiffness is characterized by increased muscle free water and GAG content. Post-treatment, the T1ρ parameters shifted toward control values. This pilot study demonstrates the application of bi-exponential T1ρ mapping as a marker for GAG content in muscle and as a potential treatment monitoring tool for patients with muscle stiffness after stroke.