The diagnosis of intermittent claudication (IC) is challenging. Imaging with radiopharmaceuticals provides a new method for detecting acute IC. Pentoxifylline improves blood flow to ischemic tissues via increasing erythrocyte elasticity and inhibiting platelet aggregation. Pentoxifylline was radio-iodinated with radioiodine-131 (131I) through a direct electrophilic substitution reaction. Furthermore, various factors that might influence the radiolabeling strategy were investigated. The radiochemical yield of [131I]iodopentoxiphyline was evaluated by using paper chromatography and HPLC methods. The biodistribution pattern of [131I]iodopentoxiphyline was studied, where Swiss albino mice was used as a model of acute limb ischemia-reperfusion. The maximum radiochemical yield of pentoxifylline was found to be 94.11 ± 2.35%. The biodistribution findings revealed that [131I]iodopentoxiphyline was significantly deposited at the ischemic site (left hind limbs), with encouraging target/non-target (T/NT) ratios. At 0.25 and 1 h post injection, the uptake of [131I]iodo-pentoxifylline was 5.30 ± 0.30 and 9.98 ± 1.12%, respectively. Also, the maximum T/NT ratio for [131I]iodo-pentoxifylline (9.45 ± 0.26) was obtained at 0.25 h post injection. Due to safety and selectivity, [131I]iodo-pentoxifylline may be a good prospective diagnostic tool for early identification of IC. Moreover, the outcome of this study can be expected to apply to I-123 as well.