BackgroundNon‐syndromic orofacial cleft (NSOC) is a complex phenotype, involving multiple genetic and environmental factors. Association studies exploring the genetic susceptibility to this prevalent oral malformation show variability of results in different populations. Using a candidate gene approach, we aimed to verify the role of four single‐nucleotide polymorphisms (SNPs) in the susceptibility to NSOC in Portuguese patients.MethodsA total of 254 non‐consanguineous individuals of Portuguese were recruited, including 120 patients with NSOC and 134 controls. About 92% of these patients had non‐syndromic cleft lip with or without cleft palate (NSCL/P) and 8% had only non‐syndromic cleft palate (NSCP). SNPs in the MTHFR (rs1801133), IRF6 (rs642961), PAX7 (rs742071) and TP63 (rs9332461) genes were studied, using a real‐time approach with TaqMan probes. Allelic, genotypic, dominant, recessive and over‐dominant models were explored using a chi‐squared test. Adjusted p‐value was calculated for multiple comparisons using the Benjamini–Hochberg false discovery rate (FDR).ResultsAll SNPs were in Hardy–Weinberg equilibrium. For MTHFR, IRF6, and PAX7 SNPs, no statistically significant difference was highlighted for any of the evaluated models. For TP63 SNP, data fitted an over‐dominant model, with a protective effect for heterozygotes (OR 1.897; CI 95% [1.144–3.147]; p < .016, when comparing controls vs. cases), but significance was lost when applying adjusted p‐value for multiple comparisons (4 × 5 tests).ConclusionIn this Portuguese population, there was no evidence of an association between the evaluated SNPs and NSOC. For TP63 SNP, the possibility of a protective effect of heterozygotes should be further investigated.