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Objectives Exclusive breastfeeding up to six months is widely recommended. Many factors may negatively impact breastfeeding, including ankyloglossia, a condition characterized by a short, thick, or tight lingual frenulum that restricts tongue movement. However, there is no validated diagnostic tool or universally accepted threshold for defining ankyloglossia. Also, evidence on its impact on early weaning, as well as on the benefits and harms of surgical intervention (frenotomy), is lacking. Nevertheless, the diagnosis and treatment of this condition are being incentivized worldwide. In Brazil, a law mandates assessing the lingual frenulum in all newborns. We are conducting an umbrella project to estimate the prevalence of ankyloglossia (prevalence study), assess its association with early weaning (cohort study), and evaluate the effects of frenotomy on early weaning (RCT). Here, our aim is to report the prevalence of ankyloglossia, according to two different thresholds, in newborns at a maternity hospital in Rio de Janeiro, Brazil.Method This study was approved by the Ethics Committee and registered with the Open Science Framework. To estimate the prevalence of ankyloglossia, a 95% confidence level, 2% precision, and an expected proportion of 10% were used, resulting in a sample of 864 mother-infant dyads. The study was carried out at a public maternity hospital in Rio de Janeiro from January to July 2024. All healthy, full-term infants weighing ≥ 2,500 g were eligible. Mothers who did not intend to breastfeed or were unable to do so due to medical recommendations were excluded. Within the first 96 hours of life, interviewers collected socioeconomic and health-related characteristics of the mother-infant dyad. Trained and calibrated examiners then assessed the lingual frenulum using the Bristol Protocol for ankyloglossia diagnosis, where scores 0–3 indicate severe ankyloglossia, 4–5 indicate suspected ankyloglossia, 6–7 are borderline, and 8 represents normal function.Results During the study period, 1,430 live births occurred, of which 398 were excluded (declined participation or did not meet inclusion criteria). Additionally, 168 eligible dyads were lost due to unexamined frenulum (dyads discharged or baby did not cry). This left 864 dyads for the final analysis. Among the infants, 52.2% were boys and most (48.6%) were white. The mean birth weight was 3,316 g (SD 391), and the mean maternal age was 27.5 years (SD 5.7). Most mothers were multiracial (45.0%), single (71.7%), multiparous (58.6%), had vaginal deliveries (70.1%), attended an average of 9.6 (SD 3.7) prenatal visits, earned up to one minimum Brazilian wage (45.3%), and had secondary school education (63.7%). The distribution of these characteristics did not differ between eligible and included dyads. The prevalence of ankyloglossia (scores 0–3) was 3.5% (95% CI 2.5–4.9), rising to 18.3% (95% CI 15.9–21) when suspected cases (scores 4–5) were included.Conclusions We found a low prevalence of severe ankyloglossia. While some suggest that suspected cases of ankyloglossia should be classified as definitive cases, there is still a lack of evidence regarding their potential negative impact on breastfeeding outcomes, particularly early weaning. To address this gap, we will follow up with the dyads to evaluate whether infants with confirmed or suspected ankyloglossia experience higher rates of early weaning compared to those without the condition. Additionally, we will randomly allocate babies diagnosed with ankyloglossia to either receive frenotomy or usual care to assess whether this intervention reduces the risk of early weaning. Ultimately, our aim is to explore whether the current diagnostic thresholds are leading to overdiagnosis and overtreatment. We hope our findings will contribute valuable insights to the ongoing debate surrounding ankyloglossia, its clinical management, and legislation regarding the screening of newborns.