The purpose of this study was to provide imaging evidence of increased glycosaminoglycan (GAG) content in patients with post-stroke muscle stiffness; and to determine the effect of hyaluronidase treatment on intramuscular GAG content. In this prospective study, we used 3D-T1ρ (T1rho) magnetic resonance (MR) mapping of the upper arm muscles to quantify GAG content in patients with post-stroke muscle stiffness before and after hyaluronidase injection treatment. For this study, healthy controls (n = 5), and patients with post-stroke muscle stiffness (n = 5) were recruited (March 2017-April 2018). T1ρ MR imaging and Dixon water-fat MR imaging of the affected upper arms were performed before and after off-label treatment with hyaluronidase injections. T1ρ mapping was done using a three-parameter non-linear mono-exponential fit. Wilcoxon Mann-Whitney test was used to compare patients' vs controls and pre- vs post-treatment conditions. The T1ρ values in the biceps were significantly higher in patients before treatment (34.04 ± 4.39 ms) compared with controls (26.70 ± 0.54 ms; P = 0.006). Significant improvement was seen in the biceps of patients before (35.48 ± 3.38 ms) and after treatment (29.45 ± 1.23 ms; P = 0.077). Dixon water-fat distribution was not significantly different in the patients compared to the controls (biceps P = 0.063; triceps P = 0.190). These results suggest that T1ρ mapping can be used to quantify GAG content in the muscles of patients with post-stroke muscle stiffness, and that muscle hyaluronan content is increased in stiff muscles compared with controls, providing imaging corroboration for the hyaluronan hypothesis of muscle stiffness.