Introduction: Little is known about the influence of biological characteristics on the occurrence of malocclusion. This study aimed to investigate the association between preterm birth and primary-dentition malocclusion and how breastfeeding and the use of pacifiers are related to this association. Methods: A representative sample (n = 1129) of children from the 2004 Pelotas, Brazil birth cohort study underwent a dental examination at age 5 years. Malocclusions were diagnosed according to the World Health Organization criteria, and the outcome was considered as the presence of moderate or severe malocclusion (MSM). Questionnaires including the children's oral health information were completed by the mothers. Data on socioeconomic status, breastfeeding, and preterm birth were obtained from previous follow-ups. Poisson regression analysis was conducted, followed by an interaction test. Results: The prevalence of MSM was 26.3% (95% confidence interval [CI], 23.6%-29.1%) in the total sample, 24.1% (95% CI, 21.5%-26.9%) in full-term births, and 42.2% (95% CI, 39.1%-45.3%) in preterm births. After adjustment, the prevalence of MSM was 42% higher in preterm births. Breastfeeding duration and pacifier use up to age 4 years modified the effect of gestational age on MSM. Conclusions: Preterm birth is associated with the development of MSM. Breastfeeding reduces the effect of preterm birth on MSM, and pacifier use strengthens this association. Dentists should be aware that preterm birth may be a risk factor for malocclusion in primary dentition. The findings reinforce the benefits of breastfeeding on occlusal development and the negative consequences of pacifier use.