OBJECTIVE. There is evidence that infants with ankyloglossia can experience breastfeeding difficulties including poor attachment to the breast, suboptimal weight gain, and maternal nipple pain, which may lead to early weaning of the infant. No studies have investigated the cause of these breastfeeding difficulties. The objective of this study was to determine the effectiveness of frenulotomy in infants experiencing persistent breastfeeding difficulties despite professional assistance by measuring changes in milk transfer and tongue movement during breastfeeding before and after frenulotomy. PATIENTS AND METHODS. Twenty-four mother-infant dyads (infant age: 33 ± 28 days) that were experiencing persistent breastfeeding difficulties despite receiving professional advice were recruited. Submental ultrasound scans (Acuson XP10) of the oral cavity were performed both before and ≥7 days after frenulotomy. Milk transfer, pain, and LATCH (latch, audible swallowing, type of nipple, comfort, and hold) scores were recorded before and after frenulotomy. Infant milk intake was measured by using the test-weigh method. RESULTS. For all of the infants, milk intake, milk-transfer rate, LATCH score, and maternal pain scores improved significantly postfrenulotomy. Two groups of infants were identified on ultrasound. One group compressed the tip of the nipple, and the other compressed the base of the nipple with the tongue. These features either resolved or lessened in all except 1 infant after frenulotomy. CONCLUSIONS. Infants with ankyloglossia experiencing persistent breastfeeding difficulties showed less compression of the nipple by the tongue postfrenulotomy, which was associated with improved breastfeeding defined as better attachment, increased milk transfer, and less maternal pain. In the assessment of breastfeeding difficulties, ankyloglossia should be considered as a potential cause.