BACKGROUND AND AIM: Ankyloglossia is a congenital anomaly that can affect breastfeeding. The aim was to evaluate the prevalence of ankyloglossia in newborns and breastfeeding difficulties reported by mothers; assess possible factors that may interfere with breastfeeding. METHODS: A cross-sectional study was conducted with 391 pairs of mothers/newborns at a university hospital. A pediatric dentist examined the oral cavity of the newborns for the occurrence of ankyloglossia. We analyzed medical records and the mothers answered a self-administered questionnaire to assess birth variables, breastfeeding difficulties, and sociodemographic factors. We calculated prevalence ratios (PRs) of breastfeeding difficulties according to the independent variables. RESULTS: The mean age of the newborns was 2.5±2.9 days and 52% were male. The prevalence of ankyloglossia was 15% and 91.4% of mothers reported not having breastfeeding difficulties. Ankyloglossia was not associated with breastfeeding difficulties (PR: 0.5; 95% CI: 0.2-1.4). Mothers with a low income (PR: 0.5; 95% CI: 0.3-0.8), those who received instructions on breastfeeding (PR: 0.4; 95% CI: 0.2-0.9), and those who breastfed exclusively (PR: 0.3; 95% CI: 0.1-0.8) had fewer breastfeeding difficulties. CONCLUSION: Successful breastfeeding was more dependent on being born at full term, the family income, receiving guidance with regard to breastfeeding, and exclusive breastfeeding. Although ankyloglossia was not associated with breastfeeding, future prospective studies should evaluate the long-term factors that may interfere with breastfeeding. RELEVANCE FOR PATIENTS: This study brings a new perspective on the importance of assessing ankyloglossia and breastfeeding difficulties, reinforces the benefits of exclusive breastfeeding and the need for breastfeeding instructions, as well as the need to evaluate breastfeeding before making a decision regarding frenectomy.