Ankyloglossia is a congenital alteration that affects the tongue's mobility, influencing craniofacial development; however, its association with malocclusion is still unclear. This systematic review and meta-analysis investigated the association between ankyloglossia and malocclusion in patients. The authors conducted a systematic review and meta-analysis of observational studies that investigated the association between malocclusion and the presence of ankyloglossia in any age group, used any assessment tool to diagnose ankyloglossia, and considered all types of malocclusion. The authors conducted searches in 8 electronic databases through July 1, 2022. They used Joanna Briggs Institute appraisal tools to assess the methodological quality and the Grading of Recommendations, Assessment, Development and Evaluations approach to assess the certainty of the evidence. Eleven studies (5 cross-sectional, 6 case-control) included 2,904 patients and over 13 occlusal alterations. The certainty of the evidence ranged from low through very low. Patients with Angle Class III malocclusion had similar or more inserted lingual frenula than patients with Class I malocclusion (standard mean difference, 0.37; 95% CI, -0.50 to 1.25) and Class II malocclusion (standard mean difference, 0.55; 95% CI, -0.52 to 1.63). Patients with Class III malocclusion had clinically significant increased mouth opening reduction compared with patients with Class I malocclusion (mean difference, 6.67; 95% CI, 4.01 to 9.33) and Class II malocclusion (mean difference, 5.04; 95% CI, 2.35 to 7.72) patients. There is uncertain evidence that ankyloglossia may be associated with the development of occlusal alterations. Ankyloglossia did not influence the Angle classification. Clinicians should closely follow children with ankyloglossia to evaluate whether this condition interferes with the occlusion. Future research should prospectively follow up on the long-term association between ankyloglossia and malocclusion. The protocol was registered a priori in the PROSPERO database (CRD42021248034). No amendments were deemed necessary after the registration of the protocol. Some subgroup analyses planned a priori were not possible, such as the severity of ankyloglossia, sex, and age, due to the lack of studies reporting data for these subgroups.