Background and Objective: The prevalence of ankyloglossia and its impact on breastfeeding practices may be overestimated, leading to surgical overtreatment in newborns. The study was conducted to estimate the prevalence of ankyloglossia in the first year of life and investigate the association with exclusive and total breastfeeding duration in different regions of Brazil. Materials and Methods: This multicenter prospective cohort study involved the recruitment of mother-infant pairs soon after childbirth in public hospitals in three state capitals in Brazil. Interviews were held with the mothers after birth, at 6 and 12 months to collect sociodemographic variables and data on exclusive and total breastfeeding duration. At 12 months of age, the children were submitted to a dental examination for classification of the lingual frenulum using the Bristol Tongue Assessment Tool. Data analysis involved Poisson regression with robust variance, with the calculation of unadjusted and adjusted relative risk (RR). Results: The final sample was composed of 293 children. The prevalence of defined and suspected ankyloglossia was 1% and 4.8%, respectively, totaling 5.8% (confidence interval [95% CI]: 3.1-8.5). No significant difference was found in the prevalence of exclusive and total breastfeeding at 1, 4, and 6 months between children with defined/suspected ankyloglossia and those without ankyloglossia. The multivariable analysis showed that the probability of the child achieving 6 months of breastfeeding did not differ between groups (RR = 0.98; 95% CI: 0.79-1.23; p = 0.907). Conclusion: The prevalence of defined ankyloglossia was very low and defined/suspected ankyloglossia was not associated with exclusive or total breastfeeding duration. Clinical Trial Registration: Registered with clinicaltrials.gov (n° NCT03841123).

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