This research communication describes the use of contrast-enhanced ultrasonographic examination (CEUS) in mammary glands of ewes for diagnosis of chronic mastitis; this is the first report of the use of this modality in diagnostic imaging of mammary glands of ruminants. For this purpose, a convex transducer was used, with the following settings: frequency: 2·0/4·0 MHz, mechanical index: 0·09, power: 22 dB, scanning depth: 70 mm, and sulphur hexafluoride in microbubbles at a dose of 20 µl as the contrast agent. In four healthy mammary glands (2 ewes), CEUS examination revealed a steady biphasic pattern of contrast agent kinetics characterised by initial uptake within 15–40 s post-injection, at which time intensity peaked with strong enhancement (130–200 AEU) followed by a gradual wash-out phase. In three mammary glands with history of clinical mastitis (2 ewes), the pattern was particularly inconsistent and unclear, with weak enhancement (<100 AEU) (P < 0·01) lasting for a short period. Notwithstanding issues regarding cost and withdrawal period of contrast-agent, this imaging modality may contribute to improved diagnosis of mastitis cases, especially on occasions when abnormalities cannot be easily confirmed by more conventional methods.