This study presents recommendations for intramuscular injection into the caudal thigh muscle of mice according to analysis of in vivo imaging of intramuscularly injected iohexol, a radiocontrast agent commonly used in CT imaging. An experienced laboratory animal technician using a Hamilton syringe intramuscularly injected iohexol into isoflurane-anesthetized female and male BALB/c mice. Injected volumes (25, 50, 100, and 200 μL) underwent CT scanning at 9 time points over a 3-h period. The distribution of the injectate in the muscles of the rear leg was examined over time for each volume group. Results indicated that 25- and 50-μL volumes remain intramuscularly. At 100 μL, mild to moderate leakage into the extramuscular tissues occurred. At 200 μL, leakage into the extramuscular tissues was moderate to severe. Our results suggest volumes of 50 μL or less are recommended for the caudal thigh muscles of mice when intramuscular pharmacokinetics are needed; volumes greater than 50 μL display variable distribution into extramuscular tissues, thus potentially yielding different pharmacokinetic profiles.