Abstract Purpose Axenfeld‐Rieger syndrome (ARS) is a rare autosomal dominant disorder that affects the anterior segment of the eye. The aim of this study was to examine the PITX2 gene to identify possible novel mutations in Pakistani and Mexican families affected by the ARS phenotype. Methods Three unrelated probands with a diagnosis of ARS were recruited for this study. Genomic DNA was isolated from the peripheral blood of the probands and their family members. Polymerase chain reaction and Sanger sequencing were used for the analysis of coding exons and the flanking intronic regions of the PITX2 gene. Bioinformatics tools and database (VarSome, Provean, and MutationTaster, SIFT, PolyPhen‐2, and HOPE) were evaluated to explore missense variants. Results We identified novel heterozygous variations in the PITX2 gene that segregated with the ARS phenotype within the families. The variant NM_153426.2(PITX2):c.226G > T or p.(Ala76Ser) and the mutation NM_153426.2(PITX2):c.455G > A or p.(Cys152Tyr) were identified in two Pakistani pedigrees, and the mutation NM_153426.2(PITX2):c.242_265del or p.(Lys81_Gln88del), segregated in a Mexican family. Conclusion Our study extends the spectrum of PITX2 mutations in individuals with ARS, enabling an improved diagnosis of this rare but serious syndrome.