Mutations in the TBX22 gene underlie an X-linked malformation syndrome with cleft palate (CP) and ankyloglossia. Its mutations also result in non-syndromic CP in some populations. To investigate whether mutations in TBX22 play a part in the formation of non-syndromic CP in the Thai population, we performed mutation analysis covering all the coding regions of the TBX22 gene in 53 unrelated Thai patients with non-syndromic CP. We identified four potentially pathogenic mutations, 359G-->A (R120Q), 452G-->T (R151L), 1166C-->A (P389Q), and 1252delG in four different patients. All mutations were not detected in at least 112 unaffected ethnic-matched control chromosomes and had never been previously reported. R120Q and R151L, found in two sporadic cases, were located in the DNA binding T-box domain. P389Q and 1252delG, found in two familial cases, were at the carboxy-terminal region, which has never been described. Our study indicates that TBX22 mutations are responsible for a significant proportion of Thai non-syndromic CP cases confirming its importance as a frequent cause of non-syndromic CP across different populations.